Protease complete mini

Protein was extracted from flash frozen cardiac muscles using T-PER (Thermo Scientific) with the addition of Protease (Complete Mini, Roche) and Phosphatase (PhosStop, Roche) inhibitors. Equal concentrations of protein were electrophoresed using Bis-Tris gels (Invitrogen) and transferred onto a nitrocellulose membrane. Membranes were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: Nox2, Catalase (Abcam), Nox4 (Novus), Nitrotyrosine (Millipore), Nix (Invitrogen), and Actin (Sigma). AT₁R and AT₂R antibodies used were purchased from Santa Cruz and previously validated for specificity in our laboratory and others [53 (link)–55 (link)]. HRP-conjugated secondary antibodies were used to detect bands (Amersham). Quantitative Western blot analyses were performed using ImageJ (National Institutes of Health).

Horseradish peroxidase (HRP)-conjugated anti-rabbit, anti-mouse immunoglobulin G, penicillin–streptomycin solution, Bradford protein assay kit, Cell counting kit-8 (CCK-8 kit), mitochondria membrane potential assay kit (JC-1), and LDH Release Assay Kit were obtained from Beyotime (Shanghai, China). Annexin V–PE/7AAD apoptosis detection kit was purchased from MULTI SCIENCES. Giemsa and crystal violet were purchased from Solarbio Bioscience and Technology (Shanghai, China). Trypan blue was obtained from Life Technologies (Carlsbad, CA, United States). BCA protein assay kit and Pierce ECL western blotting substrate were obtained from Thermo Fisher Scientific (MA, United States). Intact cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) assay kits were purchased from Seahorse Bioscience Company (North Billerica, MA, United States). Glucose was obtained from Sigma (St. Louis, MO, United States). L-glutamine was bought from Sangon Biotech (Shanghai, China). Puromycin was obtained from Amresco (WA, United States). Protease (Complete Mini) and phosphatase (PhosphoSTOP) inhibitor cocktail tablets were purchased from Roche Applied Science (Indianapolis, IN, United States). 2nbdg, EVAD, and ISRIB were gained from Selleck chemistry (TX, United States). Antibody information is shown in Supplementary Table 1.

HEK293 cells were transfected with plasmid constructs (0.6 µg/kb) expressing eIF4E or eIF4E-Myc using lipofectamine 3000 (Invitrogen). Cells were harvested 48 h post transfection and lysed with a buffer containing: 50 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% NP-40, 2 mM DTT and freshly added protease inhibitors (Roche Complete Protease Mini, #4693159001) and phosphatase inhibitors (PhosStop pellets, Sigma Aldrich, #4906845001). Cleared cell lysates were incubated with either anti-eIF4E antibody (A301-154A, Bethyl Laboratories, 1:100) or IgG on ice for 2 h. Protein G-Agarose (11719416001, Roche) was then added to each sample, and the mixture was incubated at 4 °C under rotary agitation for 4 h. Immunoprecipitates were washed with the buffer five times and eluted with the SDS-loading buffer for immunoblotting analysis.

HEK293 cells were lysed on ice for 60 min in lysis buffer containing 10 mM Tris (pH 7.4), 1% Triton X-100, 150 mM NaCl, 10% glycerol, and freshly added protease inhibitors (Roche Complete Protease Mini, Cat # 4693159001) and phosphatase inhibitors (PhosStop pellets, Sigma Aldrich, Cat # 4906845001). Cell lysates were centrifuged at 15,000 × g for 20 min at 4°C. Supernatant was collected as the soluble fraction. The pellet was washed 3 times with lysis buffer and centrifuged at 15,000 × g for 5 min each at 4°C. The pellet was resuspended in lysis buffer supplemented with 4% SDS, sonicated 3 times, boiled for 30 min, and collected as the insoluble fraction. Protein concentration was determined using Lowry Protein Assay (Bio-Rad). For Western blot analysis, proteins were subjected to 4%–12% bis-tris midi gels (Bio-Rad) and transferred to nitrocellulose membrane. Membrane was blocked with Odyssey Blocking Buffer (Fisher Scientific). The following primary antibodies were used: mouse anti-Myc (Cell Signaling, Cat # 2276, 1:1000 dilution) and mouse anti-beta tubulin (Sigma-Aldrich, Cat # T8328, 1:8000 dilution). Appropriate IRDye infrared secondary antibodies were purchased from LI-COR Biosciences and used at a dilution of 1:10000. Odyssey Infrared Imaging System (LI-COR Biosciences) was used to detect the signal of target proteins.