Protein Expression Analysis in LUAD Cells

Total protein was extracted from LUAD cells using RIPA buffer (Beyotime, Beijing, China) containing protease inhibitor and phosphatase inhibitor. The protein concentrations were detected by BCA assay (Beyotime, Beijing, China). An equal amount of protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, CA, USA). After blocking with 5% skimmed milk for 2 h, the membranes were incubated with primary antibodies specific for UBE2T (1:1000, ab140611, Abcam), FBLN5 (1:500, ab66339, Abcam), Bcl-2 (1:2000, ab182858, Abcam), Bax (1:1000, ab32503, Abcam), cleaved-caspase-3 (1:500, ab32042, Abcam), pro-caspase-3 (1:1000, ab32499, Abcam), p-ERK (1:2000, Cell Signaling Technology, #4370), ERK (1:1000, Cell Signaling Technology, #4695), p-GSK3β (1:2000, ab75814, Abcam), GSK3β (1:2000, ab32391, Abcam), β-catenin (1:1000, Cell Signaling Technology, #8480), β-actin (1:1000, ab8227, Abcam) at 4°C overnight, followed by HRP-conjugated secondary antibody (1:2000, ab205718, ab6789, Abcam) for 1 h. The positive bands were detected by using an Enhanced Chemiluminescence Kit (Thermo Fisher Scientific) [32 (link)].