Anti p16 antibody

Lysates were resolved by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA), which was incubated with the primary antibody followed by incubation with anti-rabbit, anti-mouse, or anti-goat immunoglobulin-G conjugated with horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA, USA). Specific proteins were detected by using enhanced chemiluminescence (GE Healthcare, Backinghamshire, UK). The primary antibodies for Western blotting were as follows: anti-Notch1 antibody (Santa Cruz, Dallas, TX, USA), anti-Jagged1 antibody (Santa Cruz), anti-p53 antibody (DO-1) (Santa Cruz), anti-p21 antibody (Millipore, Billerica, MA, USA), anti-p16 antibody (BD Pharmingen, San Jose, CA, USA), anti-ID1 antibody (Santa Cruz), anti-phospho p38MAPK (Thr180/Tyr182) antibody (Cell signaling, Boston, MA, USA), anti-p38MAPK antibody (Cell signaling), anti-phospho SAPK/JNK (Thr183/Tyr185) antibody (Cell Signaling), anti-JNK1/3 antibody (Santa Cruz), anti-actin antibody (Cell signaling), anti-GAPDH antibody (Santa Cruz), anti-phosphoserine antibody (Abcam, Cambridge, UK) and anti-phosphothreonine antibody (Cell signaling). To assess the phosphorylation level of Id1, cell lysates were immunoprecipitated with FLAG M2 agarose (Sigma).

SGC7901 cells were lysed with Radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing fresh protease inhibitors and PMSF. The lysates were then incubated with anti-p16 antibody (BD Pharmingen) overnight at 4 °C, followed by incubation with Protein G Plus/Protein A Agarose Suspension (Merck) for another 4 h at 4 °C. After washing 3 times with the ice-cold lysis buffer, proteins were released from the beads using SDS lysis buffer for 10 min at 95 °C and then resolved on 10 % SDS-PAGE gels and analyzed by immunoblotting. To block the nitrocellulose membranes, 5 % skimmed milk in TBST was used to reduce nonspecific background. Membranes were then incubated with primary antibodies overnight at 4 °C. After washing in TBST 3 times for 10 min each, membranes were incubated with secondary antibodies for 1 h at room temperature, and then washed again as before. Bound antibodies were detected using a chemiluminescence phototope-horseradish peroxidase kit according to the manufacturer’s instructions (Pierce, Rockford, IL, USA).