Anti p16

Proteins were extracted from the cell cultures and tissues using RIPA buffer. After centrifugation and quantification via Brad-ford assay, the proteins were loaded onto a polyacrylamide gel, and the SDS-PAGE was performed. Then, the proteins were transferred to PDVF membrane, blocked with 5% milk and incubated with the primary antibody overnight at 4°C. On the next day, the membranes were washed with TBST solution and incubated with the secondary antibody for 1 hour at room temperature. After three washes, the membranes were examined using an enhanced chemiluminescence kit. Anti-MLL3 (PLA0028, 1:1,000) and Anti-GST (SAB4200692, 1:1,000) were obtained from Sigma; anti-GAPDH (#5174, 1:1,000), anti-Flag (#14793, 1:1,000), anti-Myc (#2276, 1:1,000), anti-P16 (#80772, 1:1,000), anti-P21 (#2947, 1:1,000), anti-P27 (#3686, 1:1,000), and anti-P53 (#2524, 1:1,000) were obtained from Cell Signaling Technology. Mouse IgG (#63630, 1:2,000) and Rabbit IgG (#3900, 1:2,000) were obtained from Cell Signaling Technology.

Cells were lysed using RIPA buffer (BIOSESANG) with the Protease Inhibitor Cocktail (GenDEPOT) and EDTA (GenDEPOT). The Bradford assay (Bio-Rad protein assay reagent; Bio-Rad) was used to quantify protein concen-tration. Equivalent amounts of protein were separated using SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with 5% skim milk for 1 hour at room temperature and incubated for 24 hours at 4℃ with the following primary antibodies: anti-p16 (1：1,000; Cell Signaling Technology), anti-p21 (1：1,000; Cell Signaling Technology), anti-β-actin (1：5,000; Santa Cruz). Next, the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse antibodies (AbClon) for 1 hour at room tempe-rature. Signals were detected using the Amersham Imager 600 (GE Healthcare), and band intensities were quantified using ImageJ. β-Actin was used as the loading control. Expression was normalized to that of β-actin.

Cellular proteins were extracted with RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. Protein concentrations were measured by using a BCA protein assay kit (Thermo Fisher Scientific, USA). Protein samples were separated using 12.5% SDS-PAGE and transferred onto a polyvinylidenefluoride membrane (Millipore). Subsequently, membranes were blocked with 5% bovine serum albumin in PBS for 1 h and the appropriate antibodies at suitable concentrations were incubated overnight at 4 °C. The blots were then incubated with secondary antibodies at room temperature for 1 h. After extensive washing in TBST, protein bands were revealed with Super Singal West FemtoMaximum Sensitivity Substrate (Thermo Fisher Scientific, Rockford, USA) and visualized with Bio-Rad ChemiDocXRS system (Bio-Rad, USA). The primary antibodies were as follows: anti-GAPDH (Proteintech, 60004-1-lg), anti-p-ATM (Cell Signaling Technology, #5883), anti-p16 (Cell Signaling Technology, #80772), anti-p21 (Cell Signaling Technology, #2947), anti-cyclin E (Cell Signaling Technology, #4129), anti-cGAS (Cell Signaling Technology, #79978), anti-p-p53 (Cell Signaling Technology, #82530), anti-p-IRF3 (Cell Signaling Technology, #29047), anti-IRF3 (Cell Signaling Technology, #4302), anti-p-TBK1 (Cell Signaling Technology, #5483), and anti-TBK1 (Cell Signaling Technology, #3504).

Western blotting for Nrf2 was performed in EPCs in each group. Total proteins were prepared with RIPA lysis buffer at 4°C for 0.5 h (Beyotime Institute of Biotechnology, Haimen, China) and quantified using a BCA kit (Beyotime Institute of Biotechnology). Aliquots of cell lysates (50 μg) were separated by 10% SDS-PAGE, electrotransferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Inc.) and blocked with TBS/0.1% Tween-20 (TBS-T) buffer with 5% nonfat milk for 1 h at room temperature. Membranes were incubated with the appropriate primary antibody (anti-Nrf2, 1:1,000; cat. no. 12721 or anti-β-actin, 1:2,000, cat. no. 4970; Cell Signaling Technology, Inc., Danvers, MA, USA; anti-p16, 1:1,000, cat. no. ARG57377, Arigo Biolaboratories Corp., Taiwan, ROC) at 4°C overnight. The PVDF membranes were washed with TBS-T buffer followed by incubation with horse-radish peroxidase (HRP)-conjugated secondary antibody anti rabbit immunoglobulin G (H+L; cat. no. ANT020; Antgene Biotechnology Co., Ltd., Wuhan, China) at room temperature for 1 h. Following extensive washing, the bands were detected using a Chemiluminescence Detection System (ECL; Thermo Fisher Scientific, Inc.). More than 4 mice were included in each group.