Rabbit anti cyclin d1

Cell lysates were subjected to Western blot analysis with the following antibodies and dilutions: rabbit anti-PCGF1 (1:4000; Abcam, USA), mouse anti-β-Actin (1:4000; CWBIO, China), rabbit anti-AKT (1:4000, Abcam, USA), rabbit anti-pAKT (1:4000, Abcam, USA), rabbit anti-GSK-3β (1:4000, Abcam, USA), rabbit anti-c-Myc (1:4000, Abcam, USA), and rabbit anti-CyclinD1 (1:4000, Abcam, USA). The signals were amplified by HRP-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA), and detected via ECL Plus (Amersham Pharmacia Biotech, USA). The acquired images were then analyzed on a computer using Image J software.

Target cells were lysed on ice using radioimmunoprecipitation assay (RIPA) lysis buffer with phenylmethylsulfonyl fluoride (PMSF) and a phosphatase inhibitor cocktail, which is a serine protease inhibitor (100:1:1). The protein concentrations were determined using a bicinchoninic acid protein assay kit (Solarbo, Beijing, People’s Republic of China). The proteins (30 or 60 µg) subjected to 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blotted with primary antibodies overnight at 4°C. The following primary antibodies were used: rabbit anti-BMP7 (1:1,000), rabbit anti-Smad1/5/9 (1:1,000), rabbit anti-Snail+Slug (1:1,000), rabbit anti-cyclin D1 (1:10,000), rabbit anti-Vimentin (1:1,000), rabbit anti-N-Cadherin (1:5,000), rabbit anti-E-Cadherin (1:5,000) (all from Abcam, San Francisco, CA), rabbit anti-ID2 (1:1,000), rabbit-p-Smad1/5/9 (1:1,000) (both from CST, Boston, MD) and rabbit anti-GAPDH, mouse anti-β-Actin (1:2,000) (Proteintech, Wuhan, People’s Republic of China). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000, Proteintech). Positive labelling of the proteins on the membranes was visualized using enhanced chemiluminescence (Amersham Imager 600).