Rabbit monoclonal antibody against p62

Tissues were fixed with 10% formalin, embedded in paraffin, and sections (3 mm) were cut. Sections were deparaffinized in xylene, then rehydrated in a descending ethanol gradient. Antigen retrieval was carried out using pressure cooking in Tris/EDTA (pH 9.0), followed by incubation with 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity. Sections were washed three times with PBS for 3 min each, then incubated at room temperature for 2 h with rabbit monoclonal antibody against P62 (1:800; Abcam). Sections were again washed three times with PBS, incubated at room temperature for 20 min with biotin‐conjugated goat anti‐rabbit secondary antibody (ZSGB, Beijing, China), then stained with 3,3‐diaminobenzidine tetrahydrochloride. P62 protein expression was quantitated based on mean and integrated optical density of antibody staining.

Tissues were lysed in RIPA buffer (Solarbio, Beijing, China) containing 1 mmol/l PMSF (Solarbio, Beijing, China), and proteins were collected by centrifugation at 12,000g at 4°C for 10 min. Protein concentrations were determined using the BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of protein (20 μg) were separated by 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore, MA). Membranes were blocked for 2 h with 5% skim milk in phosphate‐buffered saline (PBS) containing Tween‐20 (PBST), and then incubated overnight at 4°C with rabbit monoclonal antibody against P62 (1:1000; Abcam, Cambridge). Actin was also detected as an internal control. After three washes in PBST for 5 min each, membranes were incubated for 2 h at room temperature with goat anti‐rabbit horseradish peroxidase‐conjugated antibody (1:5000; Abcam). Bands were visualized using an Enhanced Chemiluminescence kit (Beyotime, Shanghai, China). All experiments were carried out three times.

Total cellular protein was isolated from cardiac tissue and cardiomyocytes using a NP-40 lysis buffer (Beyotime, Shanghai, China). The protein concentrations were then assessed by a Bradford assay (Bio-Rad, Hercules, CA). After that, equal amounts of protein samples (40 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. For Western blotting, the PVDF membrane was blocked with 5% skimmed milk powder for 1 hour and then incubated with a rabbit monoclonal antibody against ATG9A (Epitomics, Burlingame, CA), a rabbit monoclonal antibody against GAPDH (Epitomics, Burlingame), a rabbit monoclonal antibody against p62 (Abcam, Cambridge, UK), or a polyclonal antibody against LC3 (Cell Signaling Technology, Danvers, MA) at 4°C overnight. On the next day, the membranes were washed with phosphate buffered saline-Tween 20 (PBS-T) thrice and then further incubated with an anti-rabbit peroxidase-conjugated secondary antibody (BOIWORLD, Wuhan, China). The immunological complexes were then visualized by chemiluminescence BeyoECL Plus (Beyotime). Protein expression was normalized to levels of GAPDH expression.