Anti akt

Immunoblot analysis was performed using standard procedures. Commercially available primary antibodies were directed against anti-phospho-c-MET (Tyr1234/1235; 1:1000; #3077; Cell Signaling Technology, Danvers, MA, USA), anti-c-MET (1:1000; #4560; Cell Signaling Technology), anti-phospho-AKT (1:1000; #4060; Cell Signaling Technology), anti-AKT (1:1000; #1085-1; Epitomics), anti-β-catenin (1:1000; #610153; BD Biosciences), anti-COX-2 (1:1000; sc1745; Santa Cruz Biotechnology), anti-β-actin (1:1000; sc47778; Santa Cruz Biotechnology), and anti-α-tubulin (1:4000; # 05–829; Millipore).

Western blot analysis was conducted using anti-phospho-AKT (ser473), anti-AKT, anti-4E-BP1 (Epitomics, Burlingame, CA, USA), anti-phospho-FOXO3a (ser253), and phospho-4E-BP1 (Ser65; Cell Signaling Technology, Danvers, MA, USA), anti-p21, anti-cyclin D1, and anti-PTEN (BD Pharmingen, San Diego, CA, USA) antibodies.

The western blot procedures used are described elsewhere 26 (link). Primary antibodies used including: anti-ITGB3, anti-MMP2 (Abcam, Cambridge, MA), anti-FAK, anti-p-FAK, anti-AKT, anti-p-AKT (Epitomics, Burlingame, CA) and anti-β-actin (Boster, Wuhan, China).

The total protein was extracted and subjected to electrophoresis. Cell lysates were prepared by using RIPA protein extraction reagent (Beyotime Biotechnology) and protein concentrations were determined by Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Samples containing equal amounts of protein were separated by 10% SDS‐PAGE and transferred onto PVDF membranes (Amersham Hybond). Membranes were blocked by 5% BSA in TBS/0.1% Tween‐20, then incubated overnight at 4°C. The expression level of each protein was detected by an enhanced chemiluminescence system (Vazyme Biotech). The primary Abs anti‐PD‐L1, anti‐UCHL1, anti‐STAT1, anti‐phospho‐STAT1, anti‐ERK1/2, anti‐phospho‐ERK1/2, anti‐P65, and anti‐phospho‐P65 were purchased from Cell Signaling Technology with catalog numbers E1L3N, D3T2E, 7649P, 9175S,4695P, 4370S, 3033T, and 8242T, respectively. The primary Abs anti‐AKT and anti‐phospho‐AKT were purchased from Epitomics with catalog numbers 1085‐S and 2214‐S, respectively. The primary Abs also included anti‐Flag (Cat# F1804; Sigma‐Aldrich) and anti‐β‐actin (Cat# PR‐0255; ZSGB‐BIO). The secondary Abs were goat anti‐rabbit IgG Ab or goat anti‐mouse IgG Ab (Cat# ZB‐2301 or Cat# ZB‐2305; ZSGB‐Bio).