Protein analysis, immunofluorescence, histology, and antibodies

For analysis of protein levels, muscles or cells were lysed directly into sample buffer (3.7 M urea, 134.6 mM Tris-HCl, pH 6.8, 5.4% sodium dodecyl sulfate (SDS), 2.3% NP-40, 4.45% β-mercaptoethanol, 4% glycerol, and 6 mg/100 ml bromophenol blue). Loading was normalized by densitometry of Coomassie-stained gels using either actin or myosin bands. Once normalized, muscle lysates were loaded onto SDS-polyacrylamide gel electrophoresis (PAGE) gels (8, 12, or 15% acrylamide concentration) or SDS-Agarose gels¹². Proteins were transferred onto nitrocellulose membranes, and detection of proteins was done using antibodies as described elsewhere⁷⁷. If not stated otherwise, biological replicates were used for immunoblot analyses. Sample sizes for quantification of protein levels are given in the figure or figure legend. Uncropped blot images for all western blot data are presented in Supplementary Data File 6.

For immunofluorescence, muscles were isolated, snap frozen while submerged in isobutane, and embedded into cryogenic molds using optimal cutting temperate (OCT) medium. Longitudinal or cross-sections of muscles were done by cryosectioning using a cryostat (Leica). Sections were collected on surface-treated microscopy glass slides (Colorfrost Plus, Fisher), dried, and stored at −80 °C until further use. Immunofluorescent staining of sections was done by fixing sections in ice-cold acetone (5 min at −20 °C), rehydration of tissue using 1× phosphate-buffered saline (PBS) (5 min), permeabilization (1× PBS, 0.2% Triton X-100; 5 min), and blocking with 1% bovine serum albumin (BSA) fraction V and 5% normal donkey serum diluted in Gold Buffer (155 mM NaCl, 2 mM EGTA, 2 mM MgCl₂, 20 mM Tris-HCl, pH 7.5) for 30 min. After blocking of non-specific binding sites, tissue sections were incubated with primary antibody diluted in Gold Buffer overnight at 4 °C. Following three washes with PBS (5 min each), sections were incubated with fluorescently labeled secondary antibodies combined with either 4′,6-diamidino-2-phenylindole (DAPI) and/or fluorescently labeled wheat germ agglutinin (WGA) (Sigma) diluted into Gold Buffer for 2 h at room temperature. After washing with 1× PBS (5 min each), sections were embedded using fluorescent mounting medium (DAKO), and covered with coverslips. Immunofluorescently labeled tissues were imaged using a Fluoview 1000 confocal microscope (Olympus), in sequential scanning mode using ×10 air or ×40 oil objectives and zoom rates between 1× and 3×. For analysis of cross-sectional areas, images were analyzed using the area-measuring tool in ImageJ.

Primary antibodies used in this study are listed in Supplementary Table 2. If not noted otherwise, all secondary antibodies were either from DAKO or Jackson ImmunoResearch. Fluorescently labeled WGA was obtained from Invitrogen/Thermo Fisher. DAPI was purchased from Sigma-Aldrich.