Ab109744

Antibodies specific to EphA2 (ab118882), YES1 (ab109744), TAZ (ab119253), and GAPDH (ab37168) and horseradish peroxidase‐couple secondary antibodies were purchased from Abcam. Antibodies targeting TEAD2 (8870), E‐cadherin (3195), N‐cadherin (13116), and vimentin (5741) were from Cell Signaling Technology. miR‐125a‐5p mimics, inhibitors (antagomir), negative controls, 3′‐untranslated region (UTR) reporter plasmids, small hairpin RNA (shRNA) specifically targeting TAZ, and control shRNA were synthesized by Ribobio Co. The sequences are shown in Table S1.

Briefly, H522 and H1975 cells were lysed by RIPA lysis (Cell Signaling Technology, Danvers, MA, USA) on ice for 30 min. After centrifuging and quantifying, 40 μg of total protein was segregated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then electrophoretically transferred nitrocellulose filter membranes (GE Healthcare, Piscataway, NJ, USA). After blocking with 3% Albumin Bovine V (Amyjet scientific, Wuhan, China), the membranes were incubated with anti-Cleaved Caspase-3 (C-Caspase3; ab2302; 1:1500 dilution; Abcam, Cambridge, MA, USA), anti-B-cell lymphoma-2 (Bcl-2; ab59348; 1:1500 dilution; Abcam), or anti-YES1 (ab109744; 1:1500 dilution; Abcam) overnight at 4°C, with anti-GAPDH (ab181602; 1:1500 dilution; Abcam) as control. After that, secondary antibody (ab1500771; 1:3000 dilution; Abcam) was added into membranes and incubated for 2 h at room temperature. The immunoreactive bands were shown on Alpha Innotech Imaging System (Protein Simple, Santa Clara, CA, USA) with ECL Western Blotting Detection Kit (Solarbio).

RIPA buffer (Beyotime) was adopted for protein extraction. Then, 30 µg total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred onto polyvinylidene fluoride membranes. Next, the membranes were experienced with 5% skim milk and then exposed to the specific primary antibodies at 4℃ overnight. The subsequent incubation at next day was performed with the secondary antibodies. The ECL kit (Beyotime) was utilized to display protein bands. The antibodies used were anti-YES1 (ab109744; 1:1,000; Abcam, Cambridge, MA, USA), anti-B-cell lymphoma-2 (Bcl-2) (ab185002; 1:1,000; Abcam), anti-Bcl-2 associated X protein (Bax) (ab32503; 1:1,000; Abcam), anti-cleaved caspase-3 (C-caspase3) (ab32042; 1:1,000; Abcam), anti-pro-caspase-3 (ab184787; 1:2,000; Abcam), anti-MMP2 (ab92536; 1:2,000; Abcam), anti-MMP9 (ab76003; 1:5,000; Abcam), anti-GAPDH (ab9485; 1:2,500; Abcam) and the secondary antibody (ab205718, 1:5,000; Abcam).