Anxa2

For Western blot analysis of total tubulin levels, ECs were seeded at 750,000 per well of a FN‐coated/BSA‐blocked six‐well plate and allowed to adhere for 90 min before being lysed in EB buffer. For the microtubule stability assay and focal adhesion enrichment, samples were prepared as above. 20 μg from each sample was loaded onto 10% polyacrylamide gels and then transferred to a nitrocellulose membrane and incubated for 1 h in 5% milk powder in PBS with 0.1% Tween 20 (PBSTw), followed by overnight incubation in primary antibody diluted 1:1,000 in 5% BSA in PBSTw at 4°C. Primaries used were against integrin beta 3 (Cell Signalling 4702), alpha‐tubulin (Abcam 7291), Gapdh (Abcam 9484), Rcc2 (Abcam 70788), Hspa1a (clone B‐6 Santa Cruz Biotechnology), Anxa2 (Abcam 41803) and Itga5 (Cell Signalling 4705). The membranes were then incubated with the appropriate horseradish peroxidase (HRP)‐conjugated secondary antibody (Dako) diluted 1:2,000 in 5% milk in PBSTw for 1 h at room temperature. The blot was visualised using Piece® ECL Western Blotting Substrate Kit (Thermo Fisher) and chemiluminescence detected on a Fujifilm LAS‐3000 darkroom (Fujifilm UK Ltd, Bedford, UK).

Cells were lysed in RIPA buffer containing fresh protease and phosphatase inhibitor cocktails (Sigma) by incubating for 20 min at 4°C. Protein concentration was determined using the BCA assay (Beyotime Biotechnology, China) according to manufacturer's instructions. Samples were then subjected to SDS-PAGE electrophoresis and transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, Billerica, MA) and incubated on a shaker overnight at 4°C with primary antibodies against KRAS (1:500, Proteintech, USA), ANXA2 (1:1000, Abcam, USA), GAPDH (1:1000, Cell Signaling Technology), Twist1 (1:1000, Abcam, USA), E-cadherin (1:500, Santa Cruz, USA) and Vimentin (1:500, Santa Cruz, USA). Horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000, Abcam) was incubated at room temperature for 1.5 h. Blots were developed using enhanced chemiluminescence detection reagents and scanned with a Molecular Imager system (Bio-Rad).