Oxyblot

Tissues were minced and homogenized, and protein gel electrophoresis samples were prepared [23 (link)]. For Western blotting, equal protein amounts (as determined by the Bradford method, Bio-Rad Laboratories, Hercules, CA) of either homogenate or immunoprecipitated samples were electrophoresed through an SDS polyacrylamide gel and then electroblotted onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked and incubated with antibodies against alpha-1 type I collagen, glutathione, nitrotyrosine, heat shock protein 90 (HSP90), cardiac sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) (Santa Cruz Biotechnology, Dallas, TX, USA), S-nitrosocysteine, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) (Alpha Diagnostics Intl. Inc., San Antonio, TX, USA), and protein carbonyls (by using OxyBlot; EMD Millipore, Billerica, MA, USA). Levels of proteins were detected using horseradish peroxidase-linked secondary antibodies (Bio-Rad) and an Enhanced Chemiluminescence System (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). SulfoBiotics Protein S-Nitrosylation Monitoring Kit (Dojindo Molecular Technologies, Rockville, MD, USA) was used to determine the number of S-nitrosylated cysteine residues.

HepG2 cells were purchased from ATCC (Rockville, MD, USA). Nile red, oleic acid, menadione (MND), trypsin and malondialdehyde (MDA) assay kit were obtained from Sigma-Aldrich (St. Louis, MO, USA), while palmitic, dihydroethidium and luminata forte chemiluminescence substrate were from Calbiochem (EMD Millipore, Billerica, MA, USA). 4′,6-diamidino-2-phenylindole, dihyrochloride (DAPI) and simple blue safe stain were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Oxy-blot, protein oxidation kit, protein G beads and anti-ATP synthase subunit-α (ATP5A) antibody were obtained from Merck (Darnstadt, Germany). Immobiline dry strip (7 cm, pH 3–10 NL) and 2D clean-up kit were purchased from GE Healthcare (Little Chalfont, UK). Other chemicals were of analytical grade and used as received.

Insulin secretion assays and immunoblotting were performed as described previously^{26 (link)}. OxyBlot assays were performed in accordance with the manufacturer’s instructions (Merck, Germany). Antibodies against BNIP3 (Cell Signaling Technology, #3769S) and α-tubulin (Sigma, #T9026) were purchased from commercial sources. Membranes were cut prior to antibody application. Specific detections of the target antigens by these antibodies were confirmed using mouse islet samples (Supplemental Figs. S5, S6).

Protein oxidation assay was performed using OxyBlot ™ (EMD Millipore, Billerica, MA, USA), following the manufacturer’s instructions. The hepatic carbonyl-containing proteins were cross-reacted with 2,4-dinitrophenylhydrazone (DNPH) to produce DNP adducts. After derivatization, equal amounts of proteins were subjected to SDS-PAGE. The oxidized proteins were analyzed by immunoblotting as described in Section 2.3.