s were transferred to si well plates, they were stimulated for 4 hours with IL 1B in serum free media. The SOCS1 overe pressing HACs were cultured in pellets 24 hours before the stimulation with IL 1B. Overe pression and knockdown of human SOCS1 To generate the pBABE viral vector containing the myc tagged human SOCS1, SOCS1 cDNA was amplified with two primer sets that con tained a BamH1 or EcoRI restriction enzyme site. PCR products were digested with BamH1 and EcoRI and cloned into the pBABE viral vectors. To produce retrovirus, the pBABE SOCS1 viral vectors were trans fected into a Platinum A retroviral packing cell line. Su pernatants were collected 72 hours after transfection. To infect SW1353 cells, viral supernatant was mi ed with fresh medium with 8 ug ml of polybrene at 1 1 ratio, and the mi ture was applied to freshly seeded cells.
To deliver SOCS1 or control shRNA into the SW1353 cells, SOCS1 shRNA or copGFP lentiviral particles were mi ed with fresh medium and 5 ug ml of polybrene, and the mi GSK-3 ture was applied to freshly seeded cells. Stable overe pressing or knockdown cell lines were selected with puromycin. To establish SOCS1 overe pressing HACs, pShuttle2 SOCS1 or empty vector was electro transfected by using a Gene Pulser cell System under the condition of 50 V and 2 ms pulse. Measurement of MMPs and TIMP 1 in culture supernatants Nontransfected and transfected SW1353 cells were plated onto 48 well plates for 24 hours and then pretreated with serum free media for 2 hours. The cells were stimulated with IL 1B for 24 hours.
The concentrations of MMP 1, 3, and ?13 and TIMP 1 in the conditioned media were measured with commercial ELISA kits according to the manufac turers instructions. Reverse transcriptase polymerase chain reaction for SOCS 1 Quantitative real time RT PCR was performed by using an ABI 7500 real time PCR machine. Specific Taqman primers and probes for SOCS1 MMP 1 MMP 3, MMP 13, ADAMTS4 were purchased from Applied Biosystems. The number fold difference in the e pression of tar get mRNA was calculated with a comparative Ct method, normalized to GAPDH. Western blotting and immunoprecipitation To prepare the total cell lysates, SW1353 cells were washed twice with ice cold PBS, harvested, and lysed in IP buffer, 150 mM NaCl, 1% Triton 100, 25 mM B glycerophosphate, phosphatase inhibitor cocktail, and protease inhibitor cocktail for 60 minutes on ice.
For immunoprecipitation, TAK1 antibody was added to the whole cell e tracts and incubated on a rotator overnight at 4 C. Then the protein G agarose beads were further incu bated for 3 hours at 4 C. The mi tures were centri fuged at 2,095 g for 3 minutes. The precipitates were washed 3 times by using pre cold IP buffer, and the beads were resuspended in 2�� SDS sample buffer. The immunoprecipitates or the whole cell lysates were separated on 10% denaturing polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were probed with appropriate primary