Anti p62

Heart tissues from study mice were homogenized and sonicated in a lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1mM EDTA, 1 mM EGTA, 1% Triton, 0.1% sodium dodecyl sulfate, and a protease inhibitor cocktail. For in vitro study, isolated cardiomyocytes from WT mice treated with paraquat or rapamycin were sonicated in the lysis buffer as described above. Myocardial protein samples were incubated with anti-Akt, anti-phosphorylated Akt (Ser⁴⁷³), anti-Atg5, anti-LC3B, anti-Beclin1, anti-p38 MAPK, anti-phosphorylated p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²), anti-Bad, anti-Bax, anti-Bcl2, anti-cleaved-caspase 3, anti-cleaved-caspase 9, anti-GRP78, anti-JNK, anti-phosphorylated JNK (Thr¹⁸³/Tyr¹⁸⁵), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control) (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-p62 (1:1000; Guinea Pig; Enzo Life Sciences, Plymouth Meeting, PA, USA), anti-IRE1α, anti-phosphorylated IRE1α (Ser724) (1:1000, Abcam, Cambridge, MA, USA), anti-GADD153 (1:1000, Santa Cruz Biotechnology, Inc. Dallas, TX, USA) antibodies. Horseradish peroxidase-coupled secondary antibodies were used for membrane incubation. After immunoblotting, the films were scanned and detected with a Bio-Rad calibrated densitometer and the intensity of immunoblot bands was normalized with corresponding band intensity of GAPDH (37 (link)).

Heart tissue was homogenized in RIPA (Millipore, Billerica MA) lysis buffer and centrifuged at 12,000 g for 20 min at 4°C. Samples containing equal amounts of protein were separated on a 10 or 12% SDS-polyacrylamide gel in a mini-gel apparatus (Mini-PROTEAN II, Bio-Rad). Proteins were then transferred to nitrocellulose membranes and subsequently blocked with 5% BSA in tris buffered saline (TBS)-tween 20. Membranes were incubated overnight at 4°C in primary antibodies: anti-PTEN (Cell Signaling, Cat#9552), anti-pAMPK (T172, Cell Signaling, Cat#2535), anti-AMPK (Cell Signaling, Cat#2532), anti-p62 (Enzo Life Sciences, Cat#GP62-C), anti-LC3B (Cell Signaling, Cat#3868S), anti-GAPDH (Cell Signaling, Cat#2118L), and anti-Pink1 (Abcam, Cat#ab23707). Blots were washed with TBS-Tween 20 and incubated with horseradish peroxidase (HRP) conjugated secondary antibody. Membranes were subsequently washed and exposed by enzymatic chemiluminescence in a GelDoc system (Bio-Rad) [25 (link)].