Anti twist

Cells (3 × 10⁵) were plated into six-well culture plates to achieve 70%–80% confluency, and were washed in PBS and suspended in 100 μL of RIPA buffer (Pierce, Dallas, TX, USA). Supernatant protein concentrations were determined using the BCA protein assay kit (Pierce). Supernatant samples containing 30 μg total protein were resolved by 10% or 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) depending on the molecular weights of the target proteins, and were transferred to immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) by electroblotting, and then probed with anti-E-cadherin (Cat# sc-21791, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-N-cadherin (Cat# sc-53488, Santa Cruz Biotechnology), anti-Vimentin (Cat# gtx100619, GeneTex, Irvine, CA, USA), anti-SNAIL1 (Cat# gtx125918, GeneTex), anti-Twist (Cat# gtx127310, GeneTex), anti-ZEB1 (Cat# gtx105278, GeneTex), anti-STC2 (Cat# sc-14352, Santa Cruz Biotechnology), anti-ANXA2 (Cat# sc-9061, Santa Cruz Biotechnology), anti-NRP1 (Cat# 3725, Cell Signaling Technology, Danvers, MA, USA), anti-SMAD2 (Cat#: 3103, Cell Signaling Technology), or anti-GAPDH (Cell Signaling Technology) antibodies. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Blots were developed using an ECL kit (Merck Millipore, Billerica, MA, USA).

A Western blot analysis was carried out as previously described^{53 (link)} using the following primary antibodies: anti-CA9 (GTX128428, GeneTex, Irvine, CA, USA), anti-GAPDH (10494-1-AP, Proteintech, Chicago, IL, USA), anti-vimentin (5741, Cell Signaling Technology), anti-Snail (3879, Cell Signaling Technology), anti-Slug (9585, Cell Signaling Technology), and anti-Twist (GTX100619, GeneTex).