Hrp conjugated goat anti rabbit

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.

The primary antibodies were optimized by use of a serial dilution of mixed human muscle standard lysate to ensure that the band signal intensities were located on the linear part of the antibody‐specific standard curve. Primary antibodies targeting the investigated Na⁺/K⁺ pump subunits and phospholemman (FXYD1) were monoclonal Na⁺/K⁺ pump α₁ (alfa6F, Developmental Study Hybridoma Bank, University of Iowa, United states of America), polyclonal α₂ (07‐674, Millipore), monoclonal β₁ (MA3‐930, Thermo Scientific), and polyclonal FXYD1 (13721‐1‐AP, Datasheet). Polyclonal MCT4 (AB3316P, Millipore) and monoclonal NHE1 (MAB3140, Chemicon) were used to detect the expression of MCT4 and NHE1. Applied antibodies targeting antioxidative and glycogen‐level‐related proteins were polyclonal SOD1 (574597, Millipore) and polyclonal SOD2 (06‐984, Millipore) (Kindly provided by Prof. H. Pilegaard, University of Copenhagen), polyclonal KAT (ab1877, Abcam), polyclonal GLUT4 (PA1‐1065, Thermo Fisher Scientific), and polyclonal GS (3893, Cell Signaling Technology). The secondary antibodies used were HRP‐conjugated goat anti‐rabbit (4010‐05, Southern Biotech), rabbit anti‐sheep (P‐0163, DAKO), and goat anti‐mouse (P‐0447, DAKO). A representative example of Western blotting is provided in Fig. 2.