Ab3191

Cells and clinical samples were lysed in RIPA buffer, and quantified by Rapid Gold BCA (Thermo, USA). Protein samples were separated equally by SDS-PAGE and electrotransferred to polyvinylidene difluoride (PVDF) membranes, which were then blocked for 1 h, and incubated with anti-DJ-1 (ab76008, Abcam, USA), anti-Bax (ab3191, Abcam, USA), anti-Bcl2 (ab196495, Abcam, USA), Anti-Cleaved Caspase-3(ab214430, Abcam, USA), Anti-CDK4(ab95255, Abcam, USA), Anti-Cyclin D1 (ab226977, Abcam, USA), Anti-E2F1 (ab137415, Abcam, USA), Anti-Cyclin D2 (ab230883, Abcam, USA), Anti-Cyclin D3 (ab112034, Abcam, USA), Anti-RB(17218-1-AP, proteintech, USA), Anti-pRb (phospho S780, ab47763,Abcam, USA), Anti-E2F1(12171-1-AP, proteintech, USA), Anti-H2A(16441-1-AP, proteintech, USA), Anti-α-Tubulin(11224-1-AP, proteintech, USA), Anti-Flag(80010-1-RR, proteintech, USA), and Anti-α-Actin(23660-1-AP, proteintech, USA). Next, the membranes were incubated in corresponding secondary antibodies for 1 h. Proteins were visualized and detected by SuperSignal West Atto(A38554, Thermo, USA).

About 50 μg protein was loaded on Any kDa™ Mini-PROTEAN® TGX™ Precast protein gels and transferred to low-autofluorescence polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). For autophagy Western blots, cells were treated with a 100 nM Bafilomycin A1 (B1793; Sigma-Aldrich St. Louis, MO, USA) 4 h before collection to accumulate LC3B in the cytoplasm. Primary antibodies were γH2A.X (1:1,000; ab11174; Abcam, Cambridge, UK), cyclin-dependent kinase 2 (Cdc2) (1:1,000; 28439S; Cell Signaling Technology, Danvers, MA, USA), cyclin B1 (1:1,000; 4138S; Cell Signaling Technology, Danvers, MA, USA), p21 (1:1,000; ab188224; Abcam, Cambridge, UK), Bax (1:1,000; ab3191; Abcam, Cambridge, UK), Bcl-2 (1:1,000; ab16904; Abcam, Cambridge, UK), caspase 3 (1:1,000; ab188224; Abcam, Cambridge, UK), SQSTM1/p62 (1:1,000; ab56416; Abcam, Cambridge, UK), LC3B (1:1,000; PM036; MBL International Corp., Woburn, MA, USA), and β-actin (1:10,000; A1978; Sigma-Aldrich St. Louis, MO, USA). Immunoblots were detected with 1:10,000 goat-anti-mouse DyLight™ 680 conjugated or donkey-anti-rabbit DyLight™ 800 conjugated using an Odyssey CLx Near-Infrared Fluorescence Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

VSMCs from each group were collected and lysed on ice using lysis buffer (Solarbio, Beijing, China). The mixture was centrifuged at 12,000 rpm at 4°C for 15 minutes. After discarding the supernatant, the protein concentration was determined using a BCA kit (Merck, Kenilworth, NJ, USA). Equal amounts of protein were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane, which was then blocked with 5% skimmed milk at 25°C. The membrane was then incubated at 4°C overnight with the following primary antibodies, which were all purchased from Abcam: anti-Bax (ab3191, 1 : 2000), anti-Bcl-2 (ab196495, 1 : 1000), anti-vascular cell adhesion molecule 1 (VCAM-1; ab174279, 1 : 2000), anti- intercellular cell adhesion molecule-1 (ICAM-1; ab109361, 1 : 2000), anti-E-selectin (ab137732, 1 : 2000), anti-GBP-1 (ab22604, 1 : 2000), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245, 1 : 5000). The next day, the membrane was rinsed and incubated with the secondary antibody at 37°C for 30 minutes. Enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific) was used to visualize the protein bands and GAPDH was used to normalize the protein levels.

T24 and 5637 cells were treated with RIPA reagents (Beyotime), and then the total protein was obtained by centrifugation (12,000 × rpm, 10 min). The protein was quantified with BCA protein assay kit (beyotime). 50 μg protein/lane was isolated in 10% SDS-PAGE, and then transferred to PVDF membrane. The membranes were sealed for 2 h at room temperature with 5% skimmed milk, and then incubated with primary antibody [xCT (1:1000, ab175186, Abcam); GPX4 (1:1000, ab40993, Abcam); Bcl-2 (1:1000, ab117115, Abcam); Bax (1:1000, ab3191, Abcam); Cytochrome C (1:1000, ab13354, Abcam); PI3K (1:1000, #4292, Cell Signaling Technology); p-PI3K p85α (1:1000, #17,366, Cell Signaling Technology); AKT (1:1000, #9272, Cell Signaling Technology); p-AKT Thr308 (1:1000, #13,038, Cell Signaling Technology); p-AKT Ser473 (1:1000, #4060, Cell Signaling Technology); mTOR (1:1000, #2972, Cell Signaling Technology); p-mTOR Ser2448 (1:1000, #5536, Cell Signaling Technology); β-actin (1:1000, ab8224, Abcam)] overnight at 4°C. Then the membranes were incubated for 2 h at room temperature with second antibody [Goat Anti-Rabbit IgG H&L (1:5000, ab96899, Abcam) or Goat Anti-Mouse IgG H&L (1:5000, ab96879, Abcam)]. The bands were visualized with chemiluminescent reagents (Beyotime) and photographed on a gel image analysis system (Bio-Rad, USA).