Acrylamide

Cells or snap-frozen aorta of $Apo{E}^{-/-}$ or $Apo{E}^{-/-}/CAV{1}^{-/-}$ mice were collected, washed with phosphate-buffered saline (PBS), and lysed with RIPA lysis buffer. Cellular lysates were centrifuged at $\mathrm{10,000}\times g$ at 4°C for 10 min. The supernatant was collected, and protein concentrations were determined with a bicinchoninic acid assay kit (Dingguo Biotechnology Co., Ltd.). Proteins ( $30\mathrm{\mu }\mathrm{g}$ ) were separated using 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with 40% acrylamide (Genview), Tris-HCl (pH 6.8 or 8.8) (Genview), 10% SDS (Genview), 10% ammonium persulfate (Macklin), and $N,N,N\prime ,N\prime$ -tetramethyl-ethylenediamine (Macklin). Subsequently, proteins were transferred onto nitrocellulose membranes (Cat. No. 40795444; Pall). The membranes were blocked with 5% bovine serum albumin (BSA) at 37°C for 2 h and then incubated with appropriate primary antibodies at 4°C overnight. After rinsing three times with Tris-buffered saline containing Tween-20 (TBST), the membranes were incubated with a secondary antibody at room temperature for 2 h. The membranes were visualized using a BeyoECL Star (Beyotime Biotechnology). Subsequently, Western blots were imaged using the Tanon 5200 system and quantified by ImageJ software (Schneider et al. 2012 (link)).