Protease and phosphatase inhibitor reagents

Clinical tissues or cultured cells were lysed with either T-PER^TM Tissue Protein Extraction Reagent (Thermo, USA) or RIPA Lysis Buffer (Thermo, USA) on ice, supplemented with protease and phosphatase inhibitor reagents (Thermo, USA). Then, 20 μg of total protein was separated and transferred to a 0.22-μm PVDF membrane (Millipore, USA). PVDF membranes were then blocked in 3–8% skim milk for 1–3 h at room temperature. Primary antibodies were incubated overnight at 4 °C; HRP-conjugated secondary antibodies were incubated for 1 h at room temperature. The protein bands were detected using chemiluminescence with imaging system (Bio-Rad, USA). An anti-GAPDH antibody (#KC-5G5, KangChen, China) was used as an internal reference; anti-Laminin-5 (γ2 chain) (MAB19562) was from Millipore (Millipore, USA); anti-histone H3 (D2B12), anti-ITGB4 (#4707), FAK Antibody Sampler Kit (#9330), anti-PI3K Kinase p110α (#4249), anti-Phospho-PI3 Kinase p85 (Tyr458)/p55(Tyr199) (#4228), anti-AKT (pan) (#4691), and anti-phospho-Akt (Ser473) (#4060) were from Cell Signaling Technology (CST, USA); anti-MMP10 antibody (ab199688) and anti-MMP13 (ab39021), antibody anti-KAT3A/CBP (ab2832), and anti-histone H3 (acetyl K27) (ab4729) were from Abcam (Abcam, UK).

Total proteins were extracted using RIPA supplemented with protease and phosphatase inhibitor reagents (Thermo-Fisher Scientific, United States). Equal amounts of protein samples were separated by SDS/PAGE and then electrophoretically transferred to PVDF membranes (Millipore, United States). Next, the membranes were probed with specific primary antibodies against p53 (dilution, 1:1,000; Abcam, United States), p21 (dilution, 1:1,000; CST, United States), RBFOX2 (dilution, 1:1,000; Bethyl, United States), E-cadherin (dilution, 1:1,000; CST, United States), N-cadherin (dilution, 1:1,000; CST, United States), vimentin (dilution, 1:1,000; CST, United States) and GAPDH (dilution, 1:5,000; Proteintech, China) at 4°C overnight, followed by incubation with HRP-conjugated secondary antibodies (dilution, 1:5,000; CST, United States) at room temperature for 1 h. Immunoreactive bands were visualized using enhanced chemiluminescence reagents (Bio-Rad, United States), and GAPDH was considered an inner loading control.

Total proteins were isolated by using RIPA supplemented with protease and phosphatase inhibitor reagents (Thermo-Fisher Scientific, Waltham, MA, USA). Nuclear and cytoplasm proteins were isolated by PARIS™ Kit (Life) according to the manufacturer’s instruction. Protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transferred proteins to PVDF membranes (Merck Millipore, Billerica, MA, USA), membranes were blocked in 5% skim milk for 1 h at room temperature followed by incubated with primary antibodies overnight at 4 °C. After three washes with TBST, PVDF membranes were incubated with HRP-conjugated goat anti-mouse (#7076, Cell signaling Technology, Danvers, MA, USA) or anti-rabbit (#7074, CST) secondary antibodies for 1 h at room temperature. Anti-GAPDH antibody (#5174), anti-Tubulin antibody (#2146), anti-Lamin B1 antibody (#13435), anti-Snail antibody (#3879), anti-E-cadherin antibody (#3195), anti-N-cadherin antibody (#13116), anti-ERK1/2 antibody (#9102), anti-Phospho-ERK1/2 antibody (#9101), anti-Raf-1 antibody (#9422) were from Cell Signaling Technology; anti-hnRNPA2B1 antibody (ab6102) was from Abcam (Cambridge, MA, USA).