Chemidoc touch detection system

Samples were lysed with RIPA containing 1% protease and phosphate inhibitor cocktail (P8340; Sigma-Aldrich). Proteins were run on 10% SDS-PAGE and then transferred to PVDF membranes, which were incubated with primary antibodies at 4°C overnight and probed by secondary antibodies. Blots were visualized with enhanced chemiluminescence (WBKLS0500; Millipore, Temecula, CA) and the ChemiDoc Touch detection system (Bio-Rad). The following primary antibodies were used: slug (9585; Cell Signaling Technology, Danvers, MA; 1:1000), β-catenin (8480; Cell Signaling Technology; 1:1000), p-eIF2α (3398; Cell Signaling Technology; 1:1000), p-GSK3β (5558; Cell Signaling Technology; 1:1000), GAPDH (ab8245; Abcam, Cambridge, England; 1:1000), C/EBPβ (ab32358; Abcam; 1:1000), N-cadherin (ab76057; Abcam; 1:1000), MMP-2 (ab92536; Abcam; 1:1000), AKT (ab8805; Abcam; 1:1000), p-AKT (ab81283; Abcam; 1:1000), vimentin (ab92547; Abcam; 1:5000), and cleaved caspase-3 (19677-1-AP; Proteintech, Rosemont, IL; 1:500).

Protein concentrations were determined using the BCA method (23225; Beyotime, Shanghai, China). Proteins (20 μg) were separated on 8–15% SDS‐PAGE and transferred onto PVDF membranes (ISEQ00010 0.22 μm; Millipore, Billerica, MA USA). Membranes were blocked for 2 h at room temperature with 5% non‐fat dry milk in TBST, incubated with primary antibodies overnight at 4°C, and probed with HRP‐conjugated secondary antibody (1:5000; Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. Proteins were visualized with enhanced chemiluminescence (Millipore) and the ChemiDoc Touch detection system (Bio‐Rad). The following primary antibodies were used: rabbit anti‐PARP, cleaved caspase‐3, microtubule associated protein 1 light chain 3 beta (LC3B), sequestosome 1 (p62), AMPK, phosphorylated‐ (p‐)AMPK (Thr172), mTOR, p‐mTOR (Ser2448), Beclin‐1, p‐Beclin‐1 (Ser93), GAPDH (Cell Signaling Technology, Danvers, MA, USA).