Anti human slug

Proteins were subjected to Western blotting as previously described.16, 17 Anti‐human HIF‐1α (1:2000; sc‐10790, Santa Cruz Biotechnology, Santa Cruz, CA, USA),18 anti‐human HIF‐2α (1:3000; NB100‐132, Novus Biologicals, Littleton, CO, USA),19 anti‐human Snail (1:2000; sc‐28199, Santa Cruz Biotechnology), anti‐human Slug (1:2000; sc‐10436, Santa Cruz Biotechnology), anti‐human Actin (1:2000; sc‐1615, Santa Cruz Biotechnology) and anti‐human Lamin B (1:3000; sc‐6217, Santa Cruz Biotechnology) antibodies were used. Then, horse radish peroxidase (HRP)‐conjugated secondary antibodies were incubated according to the manufacturer's instructions. The resultant signal was detected by chemiluminescence using the Immobilon Western Chemiluminescent HRP substrate (Merck Millipore, Billerica, MA, USA).

Proteins were extracted with RIPA lysis buffer (Beyotime Biotechnology), separated on a 10% sodium dodecyl sulfate-polyacrylamide gel (Beyotime Biotechnology), and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% dry skimmed milk, the membranes were incubated with a primary antibody at 4 °C overnight and then washed twice with Tris-buffered saline containing Tween (Haoran Biotechnology Co., Ltd., Shanghai, China). After incubation with a peroxidase-conjugated secondary antibody, the membrane was developed by enhanced chemiluminescence (Pierce, USA), following the manufacturer's instructions. Antibodies used were as follows: anti-human MST4 (rabbit monoclonal, 1:1000; Abcam, USA); anti-human p62 (mouse monoclonal, 1:1000; Santa Cruz Biotechnology, USA), anti-human Slug (mouse monoclonal, 1.1000; Santa Cruz Biotechnology, USA); anti-human LC3B (rabbit monoclonal, 1:1000; Cell Signaling Technology, USA); anti-human E-cadherin (mouse monoclonal, 1:500; Beyotime Biotechnology); anti-human β -actin (mouse monoclonal, 1:1000; Abcam).