Protease and phosphatase inhibitor cocktail

The lysates of HK-2 cells were prepared with RIPA lysis buffer containing Protease and Phosphatase Inhibitor Cocktail (Cell Signaling Technology, Boston, USA). Then the concentrations of total protein were measured by BCA method (Thermo Fisher Scientific, USA). 30 μg protein were subjected to SDS-PAGE (10%), and then transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, Beford, MA, USA). After blocking with5% bovine serum albumin, the membranes were incubated with appropriate primary antibodies at 4°C overnight, followed by incubation with HRP-conjugated secondary antibody (1:5000, Beyotime, Shanghai, China) at room temperature for 1 h. Following antibodies were used: anti-PERK (1:1000), anti-p-PERK (1:1000), anti-GPR78 (1:1000), anti-CHOP (1:1000), anti-cleaved Caspase12 (1:1000), anti-cleaved Caspase3 (1:1000) (Cell Signaling Technology, USA), anti-GAPDH (1:3000), anti-p-eIF-2α (1:1000), anti-eIF-2α (1:1000), anti-Bax (1:1000), anti-Bcl-2 (1:1000) (Abcam, Cambridge, MA, USA). GAPDH was selected as the loading control. The blots were detected using the enhanced chemi-luminescence (ECL) detection kit (KeyGen Biotech, Nanjing, China).

The frozen aortic tissues underwent homogenization through the utilization of an automated bead homogenizer. Total proteins were subsequently extracted from the aortic tissues by employing a RIPA buffer that contained a protease and phosphatase inhibitor cocktail (Cell Signaling Technology, 9806, Danvers, MA, USA). Following this, all of the protein samples for each experimental group were subjected to a 5 min heating process at 95 °C. Subsequently, equal quantities of protein were loaded and electrophoretically separated onto 12% sodium dodecyl sulfate-polyacrylamide (SDS) gels. Afterward, all proteins were transferred onto a polyvinylidene fluoride membrane. The membrane was then subjected to an overnight incubation at 4 °C with specific primary antibodies, namely SM22α (Abcam ab14106, 1:10,000), αSMA (Abcam ab124964, 1:1000), αTubulin (Abcam ab52866, 1:5000), and GAPDH (CST, 5174S, 1:5000). Following the primary antibody incubation, the membranes were exposed to the corresponding secondary antibodies. Protein bands were subsequently visualized using a chemiluminescent reagent (Thermo, 34580, Waltham, MA, USA) in conjunction with the Chem-iDoc™ Imaging System (12003153 BioRad, Hercules, CA, USA).

After the indicated stimulation, cell lysates were prepared from HAECs using a commercial cell lysis containing protease and phosphatase inhibitor cocktail (#5872, Cell Signaling Technology, USA). Protein concentration was measured using the BCA method (Thermo Fisher Scientific, USA). Protein from each group (20 μg) was subjected to 10-12% sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were then transferred to a PVDF membrane (Bio-Rad, USA), then blocked with 5 % fat skim milk in TBST. The PVDF membrane was then sequentially probed with primary antibodies overnight at 4° C and horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT. Blots were developed with enhanced chemiluminescence (ECL) western blot substrate (Thermo Fisher Scientific, USA) and exposed on X-ray film (#4741019291, Fujifilm). The following antibodies were used in this study: GPR30 (1:1000, #ab39742, Abcam, USA), KLF2 (1:1000, #ab139699, Abcam, USA), p-ERK5 (1:1000, #3371, Cell Signaling Technology, USA), β-actin (#1:10000, #4970, Cell Signaling Technology, USA), anti-rabbit IgG, HRP-linked secondary antibody (1:3000, #7074, Cell Signaling Technology, USA); anti-mouse IgG, HRP-linked antibody (1:3000, #7076, Cell Signaling Technology, USA).

Samples were lysed in a radio immunoprecipitation assay buffer (Beyotime, China) with a protease and phosphatase inhibitor cocktail (Cell Signaling Technology, United States). The protein samples were measured by a BCA protein assay kit (Beyotime, China). Fifty micrograms of liver MAM protein were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After blocking with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20, membranes were incubated with appropriate primary antibodies overnight at 4°C and then incubated with the HRP-conjugated secondary antibody for 1.5 h at room temperature. The primary antibodies were listed in Supplementary Table S1. The membranes were developed with an ECL kit (Tanon, China) using an Amersham Imager 600 (General Electric Company, United States).