RNA was quantitated as described previously[4] RNA was isolated

RNA was quantitated as described previously.[4] RNA was isolated using Trizol (Invitrogen), according to the manufacturer’s instructions. Complementary DNA was produced from RNA using the QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany). Gene-specific primer-probe sets were designed by Applied Biosystems (Foster City, CA).

We used an Applied Biosystems 7900HT Fast Real-Time polymerase chain reaction (PCR) system Cell Cycle inhibitor for quantitation of gene products. Gene expression was calculated, relative to hypoxanthine phosphoribosyltransferase, according to Pfaffl[6] and depicted as fold increase compared to FBS. Huh7.5 cells were washed extensively with OptiMEM (Gibco, Grand Island, NY) to remove albumin (ALB) present in serum. The last wash was collected to determine background levels of ALB. Cells were then kept in OptiMEM at 37°C for 6 hours, and samples were

taken every 2 hours. The amount of secreted ALB was determined using quantitative enzyme-linked immunosorbent assay (ELISA), as described previously.[4] ALB secretion (calculated as ng albumin/hour/10 × 106 cells) was normalized to FBS between experiments and expressed as fold-increase compared to FBS. Cells were grown on poly-L-lysine-coated coverslips and cultured in either FBS or HS. Lipid droplets were stained with Bodipy 493/503 (Invitrogen), according to the supplier’s instructions. Quantity of neutral lipid staining was OSI-906 visualized using a conventional fluorescence microscope (Zeiss Axiovert200; Carl Zeiss, Göttingen, Germany) and quantitated using ImageJ software (National Institutes of Health, Bethesda, MD). Images were taken using identical microscope and exposure settings. Data were collected in three independent experiments, MCE with four to eight microscopic fields per condition. Lipoprotein analysis was performed as described previously,[7] using size-exclusion chromatography (large particles elute first) combined with in-line triglyceride (TG) and cholesterol measurements. Sucrose density-gradient ultracentrifugation

was performed as previously described.[8] Fractions of 0.5 mL each were collected from the top of the gradient, and the RNA titer in each fraction was determined by quantitative reverse-transcriptase PCR (qRT-PCR). Immunoprecipitation (IP) experiments were performed as previously described.[4] Freshly collected tissue culture supernatants from infected cells were filtered through a 22-μm filter and placed in clean tissue culture plates (without cells) and kept at 37°C or 4°C. Samples were taken at the start of the incubations and then at 4- and 12-hour intervals. Viral RNA was extracted and quantitated as described above. For calculation of significance, all experiments consisted of a minimum of three independent replicates.

Given that our

Given that our HM781-36B molecular weight several reported polymorphisms (including XPC codon 939, XPD codon 751, and GSTM1 polymorphisms) were also correlated with AFB1-related HCC risk, we analyzed the relative contribution of the XRCC4 rs28383151 polymorphism after adjusting for these three polymorphisms and observed

a similar risk value. These data suggest that the rs28383151 polymorphism should be an important genetic susceptible factor of cancer risk. In addition, we found some evidence of XRCC4-GSTM1 interactive effects on HCC risk, possibly because this gene-gene interaction results in a more-obvious accumulation of DNA damage and, consequently, correlates with a higher risk for HCC. To explore the possible

pathogenesis that the rs28383151 polymorphism increases AFB1-related HCC risk, we analyzed the effects of this polymorphism on XRCC4 expression and DNA repair function. We found that rs28383151 A alleles were significantly associated with down-regulated levels of XRCC4 expression, including protein and mRNA expression. Regarding the association between rs28383151 polymorphism and DNA repair capacity, we elucidated this association using the levels of AFB1 DNA adduct and the frequency of TP53M. This was done primarily because AFB1 DNA adducts are a major type of DNA damage induced by AFB1 exposure and corresponding levels are related not only to AFB1 exposure, but also to DNA repair capacity,2, 4 whereas TP53M is the characteristic genetic change correlated with AFB1 exposure, and Linsitinib nmr higher frequency of this mutation predicts higher AFB1 exposure and higher HCC risk.2, 16 This suggests that AFB1 DNA adducts and TP53M could be regarded as biomarkers of DNA repair function related to AFB1 exposure. Our results showed

MCE that the rs28383151 polymorphism increased the level of AFB1 DNA adducts and frequency of TP53M. Together, these findings suggest that this polymorphism may decrease the DNA repair capacity of the NHEJ pathway through modulating XRCC4 expression levels and function. The DNA damage induced by AFB1 cannot be repaired effectively, with higher adduct levels leading to induction of mutations, such as in the p53 gene, and higher risk of hepatocellular carcinogenesis. Therefore, the rs28383151 polymorphism may play an important role in the carcinogenesis of Guangxiese HCC caused by AFB1. Another interesting finding of this study is that the rs28383151 polymorphism was associated with poor HCC prognosis, possibly because it correlates with the fact that this polymorphism increased the risk of PVT. Supporting our results, recent studies have shown that dysfunction of XRCC4 relates to tumor metastasis.

Given that our

Given that our Dabrafenib concentration several reported polymorphisms (including XPC codon 939, XPD codon 751, and GSTM1 polymorphisms) were also correlated with AFB1-related HCC risk, we analyzed the relative contribution of the XRCC4 rs28383151 polymorphism after adjusting for these three polymorphisms and observed

a similar risk value. These data suggest that the rs28383151 polymorphism should be an important genetic susceptible factor of cancer risk. In addition, we found some evidence of XRCC4-GSTM1 interactive effects on HCC risk, possibly because this gene-gene interaction results in a more-obvious accumulation of DNA damage and, consequently, correlates with a higher risk for HCC. To explore the possible

pathogenesis that the rs28383151 polymorphism increases AFB1-related HCC risk, we analyzed the effects of this polymorphism on XRCC4 expression and DNA repair function. We found that rs28383151 A alleles were significantly associated with down-regulated levels of XRCC4 expression, including protein and mRNA expression. Regarding the association between rs28383151 polymorphism and DNA repair capacity, we elucidated this association using the levels of AFB1 DNA adduct and the frequency of TP53M. This was done primarily because AFB1 DNA adducts are a major type of DNA damage induced by AFB1 exposure and corresponding levels are related not only to AFB1 exposure, but also to DNA repair capacity,2, 4 whereas TP53M is the characteristic genetic change correlated with AFB1 exposure, and MAPK inhibitor higher frequency of this mutation predicts higher AFB1 exposure and higher HCC risk.2, 16 This suggests that AFB1 DNA adducts and TP53M could be regarded as biomarkers of DNA repair function related to AFB1 exposure. Our results showed

上海皓元 that the rs28383151 polymorphism increased the level of AFB1 DNA adducts and frequency of TP53M. Together, these findings suggest that this polymorphism may decrease the DNA repair capacity of the NHEJ pathway through modulating XRCC4 expression levels and function. The DNA damage induced by AFB1 cannot be repaired effectively, with higher adduct levels leading to induction of mutations, such as in the p53 gene, and higher risk of hepatocellular carcinogenesis. Therefore, the rs28383151 polymorphism may play an important role in the carcinogenesis of Guangxiese HCC caused by AFB1. Another interesting finding of this study is that the rs28383151 polymorphism was associated with poor HCC prognosis, possibly because it correlates with the fact that this polymorphism increased the risk of PVT. Supporting our results, recent studies have shown that dysfunction of XRCC4 relates to tumor metastasis.

2E and Supporting Fig 5) These findings suggest increased β oxi

2E and Supporting Fig. 5). These findings suggest increased β oxidation in Thrsp-overexpressed livers. Interestingly,

expression levels and activities of glucose-6-phosphatase Selleck Afatinib (G6pase), a key gluconeogenic enzyme, were significantly increased in Thrsp-overexpressed livers (Fig. 2E and Supporting Fig. 2E), suggesting that Thrsp may increase the release of glucose from the liver and contribute to type 2 diabetes. To further confirm the lipogenic effect of Thrsp in the liver, hepatic Thrsp expression was silenced by a siRNA-based approach. As expected, liver Thrsp expression was decreased approximately 2-fold at the protein level in db/db mice with Thrsp gene knockdown (Supporting Fig. 6A). Hepatic Thrsp gene silencing significantly reduced hepatic TG content (Supporting Fig. 6B). Thrsp gene knockdown also significantly ameliorated liver steatosis of db/db mice, as evidenced by Metformin morphological changes and Oil Red O staining (Supporting Fig. 6C). Consistently, expression of FAS was also markedly reduced after Thrsp gene knockdown (Supporting Fig. 6A). In addition, coinciding with the amelioration of fatty liver, serum alanine aminotransferase

activity and hepatic interleukin-1 expression were significantly reduced in db/db mice with hepatic Thrsp gene knockdown (Supporting Fig. 7A,B), 上海皓元 suggesting a hepatoprotective effect of Thrsp knockdown on the liver. To determine the role of Thrsp in LXR-induced hepatic lipogenesis, we transfected murine primary hepatocytes with si-Thrsp or scrambled siRNA. Six hours after transfection, cells were treated with TO901317 or dimethyl sulfoxide for 36 hours. TO901317 significantly

increased the expression of Thrsp, which was abolished by si-Thrsp transfection (Supporting Fig. 8A). TO901317-induced lipid deposition in hepatocytes was significantly attenuated, but not completely abolished, by the knockdown of Thrsp (Supporting Fig. 8B). TO901317 treatment increased the expression of SREBP-1, FAS, and ACC, and Thrsp gene silencing markedly reduced ACC expression with a declining trend in SREBP-1 and FAS expression (Supporting Fig. 8C). These results imply that Thrsp mediates, at least in part, the lipogenic effects of LXR activation in hepatocytes and further supports our conclusion that Thrsp promotes hepatic lipogenesis. Because activation of the NR, LXR, also leads to enhanced lipogenesis in the liver,[8] and Thrsp expression is regulated by several NRs,[12, 13, 23] we sought to determine whether LXRs might regulate the expression of Thrsp. Mice were treated with TO901317 (5 mg/kg/day) for 3 days, and TG levels were examined. In line with previous reports,[8] TO901317 treatment resulted in significantly enlarged livers (Supporting Fig.

Specifically, an HLA class I/HCV association could either have (a

Specifically, an HLA class I/HCV association could either have (as above for class II) been reported in at least two prior studies, or it could have had a particularly strong (i.e., an odds ratio [OR] ≥3.0) relationship with HCV in one prior study. This odds ratio threshold was selected because three-fold and greater risks are considered to be strong and less

likely to be due to confounding.21 Our review identified six HLA class II alleles and three HLA class I allele groups associated with HCV viremia in two or more studies. An additional two HLA class I allele groups were strongly associated (OR >3.0) with HCV viremia PD0325901 in a single study (shown in Table 1). In contrast to HCV viremia, however, we found only three studies that examined the relation of HCV serostatus with HLA alleles in high-risk populations16–18 and there were no consistent or strong findings among these three studies. The Women’s Interagency HIV Study (WIHS) is a prospective, multicenter cohort study of HIV-seropositive (N = 2,793) and HIV-seronegative (N = 975) women enrolled through similar sources at six clinical

sites (Bronx, NY; Brooklyn, NY; Chicago, IL; Los Angeles, CA; San Francisco, CA; and Washington, DC). The initial enrollment was conducted between October 1994 and November 1995, and a subsequent second recruitment cycle occurred in 2002. Selleckchem Decitabine The recruitment methods and data collection procedures for WIHS have been described medchemexpress previously.22 Briefly, subjects in this ongoing study are evaluated every 6 months with standardized interviews, physical examination, and a blood draw. The WIHS protocol was approved by each local Institutional Review Board and all participants signed informed consent. In the current investigation we focused on WIHS women who, at the enrollment visit, had either self-reported a history of injection drug use (IDU) and were therefore considered at high risk of HCV infection and/or were HCV seropositive.

Among HCV seropositive women (N = 1,204) we limited our analysis to women with known HCV RNA status (N = 1,070), self-reported White non-Hispanic, Black non-Hispanic, or Hispanic race/ethnicity (N = 1,046), and had complete HLA data at one or more HLA loci (N = 758). Among the IDU (N = 1,161), we limited our analyses to women with known HCV serostatus (N = 1,129), self-reported White non-Hispanic, Black non-Hispanic, or Hispanic race/ethnicity (N = 1,098), and had complete HLA data at one or more HLA loci (N = 838). HCV serostatus was determined in all WIHS subjects at enrollment using a commercial second-or third-generation enzyme immunoassay. HCV viremia was determined for HCV-seropositive women using either the COBAS Amplicor Monitor 2.0, which has a linear range of 600–5.

Log-rank test was used for comparison of time-to-event curves Un

Log-rank test was used for comparison of time-to-event curves. Univariate and find more multivariate

proportional hazards models were developed to examine predictors of pretransplant mortality. Time-to-event analyses were performed on HIV-infected haemophilic and non-haemophilic transplant recipients who died (time to death), who developed graft loss (time to graft loss), or who developed organ rejection (time to rejection). Time-to-event analyses were also performed on HIV-infected haemophilic and non-haemophilic transplant candidates who died pretransplant (time to death), who underwent transplantation (time to transplant), or who developed MELD score of 25, specifically, the time to MELD = 25 from the day of study enrolment, satisfying transplant and study eligibility criteria. Among those undergoing liver transplantation, the 1-year and Selleckchem RAD001 3-year survival and 95% confidence intervals were calculated. Causes of pre and posttransplant deaths were determined, comparing co-infected haemophilic and non-haemophilic candidates. The statistical analysis was carried out using SAS version 9.2, Cary NC. All subjects provided signed informed consent in accordance with the Declaration of Helsinki. The protocol and informed consent documents were approved by the Institutional Review Board (IRB) of each institution. Of 104 HIV-HCV

enrolled candidates, nearly one-third, 34 (32.7%), underwent liver transplantation, including 7 of 15 (46.7%) with haemophilia and 27 of 89 (30.3%) without haemophilia. At baseline, as compared with non-haemophilic transplant candidates, those with haemophilia were younger (P = 0.01) and men only (P = 0.02). When the analyses were rerun, using male-only controls, results MCE公司 were similar (data not shown). The two groups did not differ in BMI (P = 0.43), CD4 + count (P = 0.48), proportion with detectable HIV RNA (P = 0.70), or detectable HCV RNA (P = 0.36), Table 1. There were also no differences in socio-economic characteristics between groups. The median duration of HCV infection among haemophilic subjects,

based on exposure in the first year of life [17], was 40 years [IQR: 33–47], whereas the median duration of HCV infection among non-haemophilic subjects, based on a conservative assumption of exposure since 15 years of age, was 32 years [IQR: 29–37], P = 0.001. Comparing the haemophilic with non-haemophilic transplant recipients, there was no difference in the median time to transplantation, 0.15 years vs. 0.03 years, respectively (P = 0.15). There was also no difference in the proportion of recipients who died after transplantation, 4 of 7 (57.1%) in haemophilic subjects vs. 14 of 27 (51.8%) in non-haemophilic subjects, (Table 2), nor in the median time to posttransplant death, 1.29 years vs. 0.75 years respectively, P = 0.64 (Fig. 1a).

It is not known, however, whether CRP is merely a marker of accom

It is not known, however, whether CRP is merely a marker of accompanying inflammation or whether it contributes causally to insulin Selleckchem Peptide 17 resistance. The objective of this study is to investigate the role that CRP may play in the development of insulin resistance. We examined the effect of single-dose intravenous

administration of purified human (h)CRP on insulin sensitivity in Sprague-Dawley rats using the euglycemic, hyperinsulinemic clamp technique. hCRP was associated with impaired insulin suppression of endogenous glucose production with no reduction in peripheral tissue glucose uptake, suggesting that hCRP mediated insulin resistance in the liver but not extrahepatic tissues. We further assessed components of the insulin signaling pathway and mitogen-activated protein kinases (MAPKs) in the liver. Liver tissues derived from hCRP-treated rats showed reduced insulin-stimulated insulin receptor substrate (IRS) tyrosine phosphorylation, IRS/phosphatidylinositol 3-kinase (PI3K) association, and Akt phosphorylation, consistent with hCRP-induced impairment of hepatic insulin signaling. Furthermore, hCRP enhanced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 MAPK as well as IRS-1 Ser612. Finally, we observed in primary cultured rat hepatocytes that U0126 (a selective inhibitor of MAPK/ERK kinase1/2) corrected

click here hCRP-induced impairment of insulin signaling. Conclusions: hCRP plays an active role in inducing hepatic insulin resistance in the rat, at least in part by activating ERK1/2, with downstream

impairment in the insulin signaling pathway. (HEPATOLOGY 2011) The appreciation that inflammation is a hallmark of the metabolic syndrome1, 2 has stimulated interest in whether systemic inflammatory 上海皓元 biomarkers such as C-reactive protein (CRP) contribute to the development of insulin resistance. The acute-phase protein CRP is a member of the pentaxin protein family involved in pattern recognition and innate immunity; it is synthesized primarily by the liver in response to inflammation.3 In addition to being an independent predictor of cardiovascular events,4 CRP is also closely associated with insulin resistance5 and related metabolic disturbances such as fatty liver disease and hyperglycemia.6, 7 It remains unclear, however, whether this association simply reflects the inflammatory milieu or whether it suggests a causative role of CRP in the progression of insulin resistance. In vitro studies have shown that human (h)CRP impairs insulin action and the insulin receptor substrate (IRS)/phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and/or activates proinflammatory pathways in various cell types.8–10 To date, in vivo data concerning the effect of hCRP on insulin action are still lacking.

With HIV/HBV-coinfected persons, there is an emphasis on using TD

With HIV/HBV-coinfected persons, there is an emphasis on using TDF (alone or part of an emtricitabine coformulation) as part of a suppressive HIV regimen because of the activity against both viruses and high threshold for HBV resistance. If tenofovir cannot be given, for example, because of renal insufficiency, entecavir can be used. Many experts recommend that 1 mg/day be used in all HCV/HIV-coinfected patients. Entecavir has some HIV activity and thus should only be used with a fully suppressive

HIV regimen.[15] This is true with regard to lamivudine and emtricitabine as well, which will select for HIV-resistant variants if used as HBV monotherapy. With HCV, the choice of ART is largely influenced by anticipated interactions with anti-HCV medications, including ribavirin (RBV) and the HCV protease inhibitors, boceprevir and find more telaprevir (Table 1A,B). In particular, zidovudine and ddl are avoided because of interactions Decitabine concentration with RBV.[16, 17] Interactions between boceprevir

or telaprevir and antiretroviral agents are complex and continue to evolve as new data become available (see below). In persons with cirrhosis, the question arises of what are the most liver-friendly ART regimens. Fortunately, most antiretroviral agents that are particularly hepatotoxic are not among the currently “recommended” agents. For example, tipranavir use is discouraged in patients with advanced liver disease because of a nearly 3-fold increase risk of liver injury.[18] In addition, the so-called “d drugs” containing deoxynucleotide analogs (didanosine and stavudine) also may increase risk of hepatic steatosis and hepatoportal sclerosis, but are no longer routinely recommended.[19, 20] Drugs, such as nevirapine, that can cause hypersensitivity reactions are also best avoided in persons with cirrhosis. In persons with decompensated (Child-Pugh class C) cirrhosis, there may be a MCE preference for avoiding some protease inhibitors

(e.g., darunavir), though others (e.g., atazanavir) may be safely administered. Natural history studies have shown that HIV coinfection promotes accelerated HCV hepatic fibrosis progression, even with excellent HIV control under ART. Moreover, in those who have progressed to cirrhosis, higher rates of liver failure and death are observed, compared with patients with HCV monoinfection.[21] The mechanisms underlying accelerated hepatic fibrosis are being increasingly understood. Though immunopathogenesis as a result of virus-specific infiltrating T cells is a key driver of liver injury, it is unlikely that the dys-regulated T-cell response in HIV coinfection can alone suffice to explain the accelerated natural history. Rather, a series of perturbations brought about by HIV infection in the liver microenvironment appears to contribute to the observed phenotype.

and Carpino et al7, 8 have proposed another new classification <

and Carpino et al.7, 8 have proposed another new classification Cisplatin order of cholangiocarcinomas based on cell lineage. Under their classification scheme, which is compatible with the pathological classification of ICC proposed by Nakanuma et al.,5 it is suggested that there are multiple cells of origin in cholangiocarcinoma, including hepatic stem/progenitor cells postulated to be located within the

canals of Hering (hepatic stem/progenitor cell lineage) or peribiliary glands (biliary tree stem/progenitor cell lineage), as well as immature or more mature cholangiocyte derivatives, that underlie biological, epidemiological, and clinical heterogeneity in small versus large duct ICCs and extrahepatic bile duct cancer. Hepatic stem/progenitor cell “biomarkers,” such as neural cell adhesion molecule (NCAM), have been demonstrated to be selectively expressed in combined HCC-CCA9 and in the bile ductular (cholangiolocellular) type.5, 10 Small bile duct type ICCs have also been suggested to originate from

interlobular bile ducts.11 Conversely, large duct or perihilar ICCs have been suggested to arise from biliary tree stem/progenitor cells or from more mature descendents.7, 8 A multistep carcinogenesis process indicative of a hyperplasia- NVP-LDE225 research buy dysplasia-carcinoma sequence is also currently recognized.12-15 In this context, malignant progression of precancerous precursor lesions, notably biliary intraepithelial neoplasia (BilIN) without12, 13 or with14 intestinal metaplasia, as well as intraductal medchemexpress papillary neoplasm of the

bile ducts exhibiting various phenotypes (e.g., intestinal type, gastric type, and oncocytic type),5 are consistent with different and distinct cell lineage pathways in the cytohistogenesis of ICC variants (i.e., conventional ICC versus less common subtypes, such as intestinal-type ICC or biliary cystic mucinous neoplasm with ovarian stroma5, 14). Although it has been generally considered that ICCs are derived from either cholangiocytes or, possibly, hepatic and/or biliary stem/progenitor cells, Fan et al.16 and Sekiya and Suzuki17 have now independently demonstrated, with unique mouse models and eloquent hepatocyte fate tracing methods, a compelling alternative to the cellular origin of ICC, namely, through transdifferentiation and neoplastic conversion of normal hepatocytes into malignant cholangiocytes by a mechanism mediated, in part, by overexpression of activated Notch. In the model described by Fan et al., ICCs induced in liver after hydrodynamic tail vein injection of the intracellular domain of Notch1 receptor plasmid, combined with concomitant injection of an Akt-overexpressing plasmid, were of the cystadenocarcinoma type, which formed in noncirrhotic liver.

Archival, formalin-fixed,

paraffin-embedded sections of l

Archival, formalin-fixed,

paraffin-embedded sections of liver specimens were obtained from the Departments of Pathology at Beth Israel Medical Center, New York, United States, Kurume University School of Medicine, Kurume, Japan, Aristotle University Medical School, Thessaloniki, Greece, and from the Liver Cancer Specimen Bank, part of the National Research Resource Bank Program, which is administered by the Korea Science and Engineering Foundation under the Ministry of Science and Technology. Approvals from the respective institutional review boards or the equivalent were obtained prior to beginning all investigations. selleck chemical The liver biopsy specimens consisted of 33 cases of chronic hepatitis B (CHB) and 69 cases of chronic hepatitis C (CHC). Histologically normal (control) liver specimens were obtained from wedge-biopsied livers of donors for liver transplantation, autopsy, or normal tissue distant from tumor in hepatic resections. The liver biopsy specimens with chronic hepatitis were staged for fibrosis according to a modified Ishak

staging system19 (1, portal fibrosis; 2, fibrous septa; 3, transition to cirrhosis; 4, established cirrhosis) and for grade of necroinflammatory activity (1, mild; 2, moderate; 3, selleck chemicals severe [i.e., with confluent necrosis]). Four-micron thick tissue sections were deparaffinized with xylene and rehydrated with graded alcohols. After washing in distilled water, sections were immersed in 3% hydrogen

peroxide to block endogenous peroxidase. Details of EpCAM staining methods used at the three institutions are given in Table 1. Other antibodies used for immunohistochemical stains included: keratin (K) 19 (clone RCK108, Dako, Glostrup, Denmark; dilution 1:20), p21WAF1/Cip1 (clone SX118, Dako; dilution 1:50), and 上海皓元 proliferating cell nuclear antigen (PCNA) (clone PC10, Dako; dilution 1:75). These stains were either performed in sequential cuts of the tissue block (EpCAM/K19) or in the same slide (double staining of EpCAM with K19, p21WAF1/Cip1, or PCNA). We used the DAKO Envision Kit (Dako) for immunohistochemistry with a single primary antibody, using 3,3-diaminobenzidine (Dako) as the chromagen. All slides were counterstained with hematoxylin. For double immunohistochemical staining, the EnVision AP system (Dako) and Vector Blue Alkaline Phosphatase Substrate Kit III (SK-5300, Vector Laboratories, Burlingame, CA) were used to detect the first primary antibody, and then the EnVision DuoFLEX Doublestain System (SK110) (Dako) and Vector NovaRED Substrate Kit (SK-4800, Vector Laboratories) were used to detect the second primary antibody.