The PCR products were ethanol-precipitated at -20°C overnight, resuspended, ligated into modified pGIR310, this website transformed into E. coli, and colonies screened. Plasmids with inserts were sequenced, and those with perfect U6 promoter and hairpin sequences were cultured, plasmids were isolated using the Qiagen HiSpeed Maxiprep Kit (Qiagen, Valencia, CA, USA), and transformed into HM1:IMSS strain trophozoites as described above. Western blotting The Igl, URE3-BP, or EhC2A shRNA transfectants were grown in 25 cm2 tissue culture flasks and selected beginning with 15 μg/ml of hygromycin, with the hygromycin level increased every 24 hours until
the final level of selection was reached, and this level was maintained for 48 hours before harvesting. The GFP control, all three Igl, and the URE3-BP
(350–378) transfectants were selected with 100 μg/ml, the URE3-BP (580–608) shRNA transfectants with 75 μg/ml, and the EhC2A samples with 90 μg/ml hygromycin. The final concentration of hygromycin selection differs since the selection was increased until the PI3K Inhibitor Library nmr desired level of knockdown was achieved. There were three biological replicates per shRNA transfectant, and one for the HM1:IMSS nontransfected trophozoites. Trophozoites were harvested as described above for transfection, counted, resuspended in ice cold Lysis Buffer (150 mM NaCl, 50 mM Tris, 5× Sigma protease inhibitor cocktail (P2714) 4EGI-1 supplier (Sigma-Aldrich, St. Louis, MO, USA), 25 μg/ml E-64 (Sigma-Aldrich, St. Louis, MO, USA)) at an initial concentration of 2 × 106–5 × 106 amebae/ml, and lysed by sonication by pulsing twice for 10 seconds each with a 10 second rest on ice between pulses. Protein was quantified and sample
lysates were diluted to the same protein concentration, were serially-diluted 1:2, 1:4, and Gemcitabine 1:8 with Lysis Buffer, and were subjected to SDS-PAGE on 12% (Igl) or 15% (URE-BP and EhC2A) gels. All sample lysates and dilutions were done in triplicate (technical replicates). Gels were transferred to PVDF membrane, membranes were cut in half so each half could be probed separately, were blocked in 5% milk, and incubated with either antibodies against Igl1, URE3-BP, EhC2A, or control antibodies against actin (anti-actin from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA) or Sigma (Sigma-Aldrich, St. Louis, MO, USA)). The ECL kit from Roche (Roche Applied Science, Indianapolis, IN, USA) was used to treat membranes after secondary antibody incubation, bands were visualized on film, film images were electronically scanned, and Scion Image Beta 4.0.3 software (Scion Corporation, Frederick, MD, USA) was used to quantify band intensity.