Rabbit anti p16

Cell or tissue lysates were extracted for loading into 10% SDS-PAGE gels and immunoblotting was performed as previously described 34 (link). Primary antibodies, including rabbit anti-Cyp27b1/1α(OH)ase (Abcam, ab206655), rabbit anti-VDR (Abcam, ab3508), rabbit anti-Sirt1 (Millipore, 07-131), rabbit anti-p16 (Proteintech, 10883-1-AP) and rabbit anti-β-actin (Cell Signaling Technology, 8457S) were used for immunoblotting. Immunoreactive bands were visualized with ECL chemiluminescence (Bio-Rad) and analyzed by Image J.

The RIPA (Sigma-Aldrich) buffer containing complete Protease Inhibitor Cocktail (Sigma-Aldrich) and PMSF (Beyotime, China) was used to extract proteins from lung tissues and cells. The extracts were centrifuged at 4℃ for 15 min at 12,000 rpm and the supernatants were quantified with the BCA protein assay kit (Beyotime). Equal amounts of total protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, USA). Membranes were blocked with 5% skim milk at room temperature for 1 h and then incubated with the primary antibodies at 4℃ overnight. After washing with TBST (Tris-buffered saline, 0.1% Tween-20), the membranes were subsequently incubated with secondary antibodies for 1 h at room temperature. Protein bands were visualized with the Enhanced Chemiluminescence Detection Kit (Thermo Fisher) via the Tannon luminescent imaging system. Image J was used for the quantitative analysis of the protein bands. The antibodies used are listed below: rabbit-anti-Bmi-1(CST, 1:2000 dilution), rabbit-anti-p16 (Proteintech, USA, 1:1000), mouse-anti-β-actin (CST, 1:5000 dilution), rabbit-anti-GAPDH (CST, 1:5000 dilution), rabbit-anti-pH2A.X (Beyotime, 1:1000).[33 (link)].