Ab47365

HTR-8/SVneo and JAR cells transfected with plasmid vectors or siRNAs were lysed to extract total proteins in the radioimmunoprecipitation assay lysis buffer (#C500005; Sangon Biotech) supplemented with protease inhibitors. Protein concentrations in each sample were determined by Bradford assay. Then, 25 µg of protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and loaded onto polyvinylidene fluoride membranes (#F019531; Sangon Biotech) in transfer buffer. Next, the membranes were blocked with 5% nonfat milk diluted in tris-buffered saline solution containing 0.5% Tween-20 at 4 °C overnight, and then incubated with the following primary antibodies (Abcam, Cambridge, UK) at 4 °C overnight: anti-SRSF1 (ab38017; 1:1000), anti-cleaved-caspase-3 (ab2302; 1:500), anti-Bcl-2 (ab59348; 1:500), anti-pmTOR (ab84400; 1:900;), anti-p4EBP1 (1:800; ab47365), and anti-β-tubulin (ab6046; 1:500). Finally, the membranes were cultured with horseradish peroxidase-labeled secondary antibodies immunoglobulin G (IgG) for 1 h at room temperature, followed by the detection with ECL analyses kits (#D601039; Sangon Biotech). All experiments were repeated at least three times.