Anti beta actin antibody ab8226

Wild‐type and mutant (G306R) plasmids were transiently transfected into HEK293T (human embryonic kidney) cells cultured in Dulbecco's Modified Eagle's Medium (DMEM), using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Total protein extracts (20 μg) from cell lysates were electrophoretically separated on 10% denaturing polyacrylamide gels and then transferred to Polyvinylidene difluoride (PVDF) membranes (Roche, Basilea, Switzerland). A high range SigmaMarker (Sigma‐Aldrich, Saint Louis, MO; molecular weight range, 36 000‐200 000 kDa) was used to characterize protein migration. Membranes were blocked in Tris‐buffered saline with 0.1% Tween 20 containing 5% non‐fat dried milk and reacted overnight at 4°C with the primary monoclonal antibody, anti‐FLAG mouse M2 clone (Sigma‐Aldrich) diluted 1:2000, and anti‐beta Actin antibody ab8226 (Abcam, Cambridge, UK), diluted 1:1000. After rinsing, filters were incubated with horseradish peroxidase‐conjugated goat anti‐mouse IgG secondary antibody (Santa Cruz), diluted 1:10000. Signals were developed using the enhanced chemiluminescent horseradish peroxidase (HRP) substrate Westar ETA C ultra (Cyanagen, Bologna, Italy), and specific protein bands were revealed using the G:BOX Chemi XT4 gel doc system (Syngene, Frederick, MD).

Anti-CUL4B antibody (ab227724), Anti-beta Catenin antibody (ab32572), Anti-c-Myc antibody (ab32072), Anti-Cyclin D1 antibody (ab16663), Anti-MMP3 antibody (ab52915), Anti-Fibronectin antibody (ab2413) and Anti-beta Actin antibody (ab8226) were purchased from Abcam (Waltham, MA, United States). miR-942-5p mimics, inhibitors and negative control (miRNA NC) were purchased from Shanghai GenaPharma Pharmaceutical Technology Co., Ltd. (Shanghai, China). CUL4B, β-catenin, MMP3, FIBRONECTIN, c-Myc, CCND1 and β-ACTIN primers were produced by Shanghai Sangon Biological and Technological Company (Shanghai, China). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazoliumbromide] and DMSO (dimethyl sulfoxide) were purchased from Sigma Inc.(St. Louis, MO, United States).