Mouse anti cdk4

Cells transduced with shRNA-expressing lentivirus were divided into two groups (shCon, shZFR). PANC-1 cells were harvested after lentivirus transduction for 6 days. Total protein was extracted with 2 × SDS Sample Buffer [100 mM Tris–HCl (pH 6.8), 10 mM EDTA, 4 % SDS, 10 % Glycine]. Total protein concentration was determined by BCA assay. Protein extracts were separated by 10 % SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5 % nonfat dry milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at 37 °C, and then incubated overnight at 4 °C in TBST with primary antibody, including rabbit anti-ZFR (1:1000, SAB2104153, sigma), mouse anti-CDK4 (1:500, #2906, cell signaling), rabbit anti-CDK2 (1:1000, #2546, cell signaling) and rabbit anti-GAPDH (1:100000, 10494-1-AP, Santa cruz). Following incubation with horseradish peroxidase-conjugated goat anti-rabbit/mouse secondary antibody for 1 h, the membranes were detected using enhanced chemiluminescence (ECL) kit (Amersham) and visualized by exposure to X-ray film. GAPDH was used as a control to verify equal protein loading.

All sample protein extracts were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking for 2 h in Tris-buffered saline with 0.1% Tween^® 20 detergent (TBST) containing 5% nonfat milk, the membranes were incubated with primary antibodies diluted in TBST containing 5% nonfat milk at 4 °C overnight. The following primary antibodies were used: mouse anti-glyceraldehyde 3-phosphate dehydrogenase (PMK Biotechnology, Shijiazhuang City, China); mouse anti-cyclinD1 (Cell Signaling Technology, Danvers, MA, USA); mouse anti-CDK4 (Cell Signaling Technology); mouse anti-P21 (Cell Signaling Technology); mouse anti-Bax (Cell Signaling Technology); and mouse anti-caspase-3 (Abcam, Shanghai, China). The membranes were washed 3 times with TBST and incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. They were then washed 3 times with TBST and the signals were visualized using ECL plus reagents (Amersham Biosciences, Buckingham, UK).