Sqstm1 p62 monoclonal antibody

We used the LC3B monoclonal antibody (Cell Signaling, Danvers, MA, USA; #3868) at 1:1000 dilution, SQSTM1/p62 monoclonal antibody (Cell Signaling, Danvers, MA, USA; #5114) at 1:1000 dilution, and a beta-Actin antibody (Sigma, St Louis, MO, USA; #A5441) at 1:5000 dilution for the quantitative Western blotting analysis as described by Xie et al. [44 (link)]. We analyzed the signal intensity using the Quantity One image analysis program (Bio-Rad, Hercules, CA, USA). We used beta-Actin to standardize the amounts of protein (e.g., calculating the ratio of the amount of LC3-II to the amount of beta-Actin) and to limit disparities in the quantity of protein loaded. We expressed the protein levels as a percentage when comparing them to those in the control condition.

Cultured cells and fragments of the arterial wall were extracted by RIPA Lysis Buffer (Beyotime) containing protease and phosphatase inhibitors. Quantified samples with 30 μg total protein were separated on 12% SDS-ployacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Wattford, UK), which were blocked with 2% (wt/vol) bovine serum albumin in Tris-buffered saline solution containing 0.1% Tween-20 for 1 h. Membranes were incubated overnight at 4 °C with the primary antibodies for LC3 (1:1000 dilution; 2775 S) and SQSTM1/p62 monoclonal antibody (1:1000 dilution; 8025 S, both from Cell Signaling Technology), mouse monoclonal antibody against ACTIN (1:2000 dilution; sc-7210, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GAPDH, followed by peroxidase-conjugated secondary antibody (A0208, Beyotime) for 1 h at room temperature. After washing, signals were visualized using Super Signal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL, USA). Western blots were prepared at least three times for each sample.