Csb pa025358la01hu

After 72 h of transfection, cells were added with an appropriate amount of RIPA (PMSF) buffer. Then, the total protein was extracted by centrifugation, the total protein concentration was determined using the BCA method, and the protein was separated using SDS-PAGE. The proteins were electrotransferred to PVDF membranes and blocked with 5% milk blocking solution for 1 h to block nonspecific bindings. The membranes were incubated with the following primary antibodies overnight at 4 °C and washed three times with 0.05% TBST for 5 min each time: CXCL11 (CSB-PA06119A0Rb; Cusabio, Wuhan, China), Vimentin (10366-1-AP; Proteintech, Wuhan, China), Twist (CSB-PA025358LA01HU, Cusabio), β-actin (60008-1-Ig, Proteintech), IFIT1 (CSB-PA011018LA01HU, Cusabio), and IFIT3 (CSB-PA011022HA01HU, Cusabio). The membranes were then incubated with secondary antibodies (HRP-labeled goat anti-rabbit IgG and goat anti-mouse IgG) at room temperature for 1 h and washed three times with 0.05% TBST for 5 min each time. The signal was detected by the ECL chemiluminescence method.

RIPA buffer (Catalog number: P0013C; Beyotime) and protease inhibitors were used to obtain total protein from either target tissues or transfected cells or untransfected target cells. The protein concentration was established by the BCA quantitative method (Catalog number: P0012, Beyotime). Then the protein samples were loaded into SDS‐PAGE (8%–15%) for separation and transferred to PVDF membrane. By incubating the membrane with a 5% milk blocking solution for 1 h, non‐specific binding prevention could be achieved. Then the membrane was incubated with anti‐CERCAM (1:500, 16411–1‐AP; Proteintech), anti‐PCNA (1:2000, 10205–2‐AP, Proteintech), anti‐Vimentin (1:2000, 10366–1‐AP, Proteintech), anti‐Twist (1:500, CSB‐PA025358LA01HU; Cusabio), anti‐N‐cadherin (1:2000, 22018–1‐AP, Proteintech), anti‐E‐cadherin (1:5000, 20874–1‐AP, Proteintech), anti‐Akt (Y409094, Abm), anti‐p‐Akt (1:500, Y011054, Phospho‐ser473; Abm), or anti‐β‐actin (1:5000, 60008–1‐Ig, Proteintech) at 4°C for 24 h, and then incubated with the matching secondary antibody at 37°C for 1 h. Enhanced chemiluminescence (ECL) reagents and Automatic chemiluminescence imaging system (Tanon 5200) were used to visualize proteins. Experiments were repeated for 3 times.