Ab40742

Cell death was quantified by measuring LDH release to the supernatant, using the LDH cytotoxicity detection kit (Takara, Clontech). To normalize for spontaneous cell lysis, the percentage of cell death was calculated as follows: (LDH_sample – LDH_{negative control})/(LDH_{positive control} – LDH_{negative control}) × 100. PI influx measurement was performed as previously described^{46 (link)}. The levels of IL-18 were measured by ELISA (R&D Sytems), according to the manufacturer’s instructions. For western blotting analysis, cell lysates were prepared and supernatants were precipitated. Mouse anti-caspase-4 4B9 (ADI-AAM-114-E, Enzo Life Sciences, 1:750), rabbit anti-GSDMD (ab210070, abcam, 1:1000), rabbit anti-GBP1 (ab121039, abcam, 1:1000), mouse anti-GAPDH (AM4300, Thermo Scientific, 1:1000), mouse anti-V5 (R960-25, Thermo Scientific, 1:2000), mouse anti-GFP (632381, Clonetech, clone JL-8, 1:5000), mouse anti-HA (ENZ-ABS-118-0200, Enzo Life Sciences, 1:2000), mouse anti-tubulin (ab40742, Abcam, 1:2000) were used and detected with horseradish peroxidase-conjugated secondary antibodies (1:5000, Southern Biotech).

Cardiac muscle (25–50 mg) was homogenized as previously reported [22 (link)] and 5μg of homogenate loaded for SDS-PAGE. Proteins were separated on a 6%, 7.5%, 10% or 12% resolving gel as required to optimize for MW separation, and transferred to polyvinylidene difluoride membrane (Roche, Laval, QC, CA). The following commercially available antibodies were used: total OXPHOS antibody cocktail (Abcam, Cambridge, MA, USA, ab110413, 1:500,), eNOS (Abcam, ab5589, 1:1000), VEGF (Abcam, ab46154, 1:1000), HIF1α (Abcam, ab463, 1:1000), alpha tubulin (Abcam, ab40742, 1:5000), muscle RING finger protein-1 (MuRF1; Santa Cruz Biotechnology, Dallas, TX, USA, sc-32920, 1:500), Muscle atrophy F-box (MAFbx; Santa Cruz, sc33782, 1:500), forkhead transcription factor-3a, Serine residue 253 (FOXO3a Ser253; Abcam, ab47285, 1:500), atrial natriuretic peptide (ANP; Abcam, ab180649, 1:500), BNP (Abcam, ab19645, 1:500) and beta-myosin heavy chain (β-MHC; Abcam, ab172967, 1:2000). All samples were detected from the same Western blot by cutting gels and transferring onto a single membrane to limit variability. Equal loading of protein was verified using Ponceau staining as well as constant alpha tubulin. All blots were quantified using enhanced chemiluminescence (Perkin Elmer, Woodbridge, ON, CA) and quantified by densitometry (Alpha Innotech Fluorchem HD2, Fisher Scientific, Ottawa, ON, CA).

For western blotting analysis, cells were lysed in 66 mM Tris-HCl pH 7.4, 2% SDS, 10 mM DTT, NuPage LDS sample buffer (Thermo Fisher). When mentioned, supernatants were precipitated with methanol and chloroform, using standard methods, and combined with the cell lysates. Proteins were separated on 4–15% polyacrylamide gels (Bio-Rad) and transferred onto nitrocellulose membrane using Transblot Turbo (Bio-Rad). The antibodies used were: anti-mouse GSDMD (EPR19828; Abcam; 1:1,000), anti-mouse NINJ1 monoclonal antibody (rabbit IgG2b clone 25; a gift from Genentech; 1:8,000), anti-human caspase-4 (ADI-AAM-114-E; Enzo Life Sciences; 1:1,000), anti-V5 (46-0705; Invitrogen; 1:2,000) and mouse anti-tubulin (ab40742; Abcam; 1:2,000). Purified human NINJ1 was detected using mouse anti-human NINJ1 (610777; BD Transduction Laboratories; 1:1,000). Primary antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (4030-05; Southern Biotech; 1:5,000), HRP-conjugated goat anti-mouse (1034-05; Southern Biotech; 1:5,000 or 12–349; MilliporeSigma; 1:2,000) secondary antibodies.

Subcellular fractionation was performed with a PARIS™ kit (#AM1921, Life Technologies) as previously described [27 ]. Equal amounts of RNA from each fraction were subjected to RT‐qPCR, and the results were calculated using the comparative Ct method 2‐(ΔCt) and shown as a percentage, considering the total quantity of RNA recovered from each fraction. To verify the nuclear and cytoplasmic fractionation of the mRNA, RNU6B and GAPDH were used as controls, respectively. The separation was confirmed at the protein level by western blot with HISTONE H3 (#ab1791, Abcam, Cambridge, UK, 1 : 5000) and α‐TUBULIN HRP (#ab40742, Abcam, 1 : 5000).