Anti β actin ab

Spleen cells were isolated from the spleens of B6 mice and suspended in RPMI‐1640 (Wako) supplemented with 10% FBS and 50 μmol/L 2‐ME (Sigma‐Aldrich). They were plated at 1 × 10⁷ cells per well in 2‐mL 6‐well flat‐bottomed tissue culture plates coated with anti‐CD3 mAb (Clone 17A2, 1 μg/mL; Becton Dickinson). Soluble anti‐CD28mAb (Clone 37.51, 1 μg/mL; BioXCell) was also added. The spleen cells were cultured in an incubator at 37°C under 5% CO₂ and 95% air for 2 days, and then subjected to western blotting.
The cells were treated with RIPA lysis buffer (Wako) containing phosphatase inhibitor cocktail and protease inhibitor cocktail from different groups at the indicated time points. Proteins were subjected to electrophoresis and transferred onto PVDF membranes (Bio‐Rad). The PVDF membranes were blocked with 3% skin milk for 1 hour at room temperature, and then incubated with the primary Abs overnight at 4°C. Appropriate HRP‐conjugated secondary Abs were applied for 1 hour at room temperature. Proteins were extracted and blotted with anti‐β‐actin Ab (1:4000; Abcam), anti‐Sirt1 Ab (1:1000; Abcam), and total OXPHOS Rodent Ab (Abcam). The proteins were detected with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The relative optical density of each specific band obtained was measured using the ImageJ software program (NIH).

Western blot was performed as reported previously.21 (link) Briefly, the ASMCs were lysed with the RIPA buffer, adding phosphatase and protease inhibitors (Beyotime, Shanghai, China). The concentration of the proteins was tested with the Bicinchoninic Acid Kit (Thermo Scientific, Rockford, USA). The primary antibodies used in this part were anti-Cdk6 Ab, anti-pRb (Cell Signaling, Inc. Boston, USA), and anti-β-actin Ab (Abcam, Cambridge, UK). The relative level of proteins was measured with the Image J Software (National Institutes of Health, Bethesda, USA).