Rabbit polyclonal anti p21

All primary and secondary antibodies were diluted in PBS Tween-20 0.1% containing BSA 2%. Primary antibodies used were: rabbit polyclonal anti-HADHA (Proteintech, Cat# 10758-1-ap) 1:500; rabbit polyclonal anti-SQSTM1/p62 (Novus, Cat# NBP-48320, Centennial, CO, USA) 1:4000; mouse monoclonal anti-phosphoERK1/2 (Santa Cruz, Cat# 7383, Dallas, TX, USA) 1:500; rabbit polyclonal anti-ERK1 and anti-ERK2 (Santa Cruz, Cat# sc-93 and Cat# sc-154) 1:500; rabbit polyclonal anti-p21 (Proteintech, Cat# 10355-1-AP) 1:800; rabbit polyclonal anti-LC3 I/II (Novus, Cat# NB100-2220) 1:1000; rabbit monoclonal anti-phospho4EBP1 (Cell Signaling Technology, Cat# 2855T, Danvers, MA, USA) 1:500; mouse monoclonal anti-4EBP1 (Proteintech, Cat# 60246-1-1g) 1:500; mouse monoclonal anti-β Actina (Sigma Aldrich, Cat# A5441) 1:10,000.
Goat polyclonal anti-mouse 1:15,000 (Santa Cruz, Cat# sc-2005) and anti-rabbit 1:15,000 (Santa Cruz, Cat# sc-2004) were used as secondary antibodies.

For Western blot analysis, HUVEC lysates were collected with RIPA Buffer (Beyotime, Shanghai, China) and protein concentration was determined using a BCA protein assay kit (Thermo Fisher, Rockford, IL, USA). Proteins were separated by electrophoresis with 10%-12% SDS polyacrylamide gel and then were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Following blocking at room temperature with 5% skimmed milk for 1 h, the membranes were incubated with the indicated primary antibodies at 4°C overnight. After that, membranes were washed in Tris-buffered saline tween (TBST) and latter incubated with secondary anti-rabbit or anti-mouse antibody. The bound secondary antibody was visualized by chemiluminescence using ECL™ Western Blotting Detection Reagent (GE Healthcare). The rabbit polyclonal anti-CPT1A, rabbit polyclonal anti-p53, and rabbit polyclonal anti-p21 were obtained from Proteintech Group (IL, USA), and the rabbit polyclonal anti-p16 was purchased from Abcam (Cambridge, UK).