Rabbit anti p27

The VSMC and VEC cultures were prepared from 6 rats each. Cells were seeded at a density of 4 × 10⁴/ml on coverslips in 6 well plates which were pre-incubated with 0.1 mg/ml polylysine in PBS for 30 min at 37°C. The coverslips were removed at the pre-designed time points with sterile tweezers, washed with sterile PBS three times, fixed with methanol for 15 min, and incubated with antibody blocking solution for 8 hours at 4°C. The coverslips were then incubated with 1:1000 dilution of rabbit anti-p27, or a 1:500 dilution of goat anti-EGFP (all from Abcam) for 8 hours at 4°C. After that, the coverslips were incubated with a 1:1000 dilution of Cy3-linked anti-rabbit antibody, or a 1:1000 dilution of FITC linked anti-goat antibody (all from Jackson) for 1 hour at 37°C, and then incubated with DAPI (0.5 μg/ml, Sigma) for 5 min at 37°C. The coverslips were visualized with an Olympus, BX51-type fluorescence microscope. Each intervention group was done in triplicate.

The primary antibodies used in this study included the rabbit anti-Atg5 antibody (Cell signaling, cat:12994), rabbit anti-LC-3 antibody (MBL, cat: PM036), rabbit anti-p62 antibody (Cell Signaling, cat:5114), rabbit anti-actin antibody (Abcam, cat: ab8227), rabbit anti-Beclin-1 antibody (Abcam. ab51031), rabbit anti-Cyclin-D1 (Cell Signaling, cat:2978), rabbit anti-Cyclin-A2 (Cell Signaling, cat:4656), rabbit anti-Cyclin-E1 (Cell Signaling, cat:4129), rabbit anti-Cyclin-B1 (Cell Signaling, cat:4138), rabbit anti-p53 (Cell Signaling, cat:2524), rabbit anti-p21 (Abcam, ab7960), rabbit anti-p27 (Abcam, ab7961), rabbit anti-Caspase 8 (Cell Signaling, cat:9496), rabbit anti-Caspase 9 (Cell Signaling, cat:9509), rabbit anti-Caspase 3 (Cell Signaling, cat:9662), rabbit anti-Cytochrom C (Cell Signaling, cat:4272), rabbit anti-COX IV (Cell Signaling, cat:4844).