Western chemiluminescent hrp substrate ecl

The human tissue specimens, mouse aorta tissue specimens, and cultured VSMCs were homogenized in Radio-Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime, China) on ice and centrifuged at 12,000 rpm. Proteins were separated using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. The membrane was incubated with an anti-CA1 antibody (Abcam, USA, catalog number: 108367) at 4°C overnight. The membrane was then incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody at 37°C for 1 h. The membrane was developed using Western Chemiluminescent HRP Substrate (ECL, Millipore). The expression level of GAPDH was used as an internal control. The grayscale value of the target protein was quantified using ImageJ software (National Institutes of Health, USA).

Cells were washed twice with PBS and lysed in RIPA Lysis Buffer (Millipore, Billerica, MA, USA) on ice for 10 min. After spin at 12,000 rpm for 15 min at 4°C, the supernatants were collected. The concentrations of proteins in supernatants were determined by BCA assay. SDS-PAGE sample loading buffer (Biyuntian, Shanghai, China) was added into the same amount of protein from different samples and boiled for 5 min. The proteins were separated by SDS-PAGE gel and then processed for immunoblotting. SDS-PAGE gel was transferred to PVDF membrane (Millipore, Billerica, MA, USA) followed by being blocked in TBST with 5% non-fat dry milk. Antibodies were used as follows: phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (1:1000, Cell Signaling Technology) and HRP-conjugated goat anti-rabbit IgG secondary antibody (1:2000, Abcam). Protein bands were detected by Western Chemiluminescent HRP Substrate (ECL) (Millipore) and visualized by ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA).