SDS gel electrophoresis and Western blotting

SDS-agarose (SDS-AGE) gel electrophoresis and Western blot experiments have been previously described (Greaser and Warren, 2009; van der Pijl et al., 2018). Titin migration distance and bandwidth (full width at half maximum; FWHM) were determined (see Fig. S5 for an illustration of the analysis). Procedures and antibodies used are described in the text at the bottom of the PDF and Table S4.

Titin isoform size and splice variation in human diaphragm biopsies. Control (n = 16) and critically ill (n = 21) patient samples were migrated on SDS-AGE gels; bandwidth was determined by generating plot-profiles fitted with Gaussian fits to generate FWHM for titin and myosin heavy chain (MyHC) bands. (A–C) FWHM of titin/MyHC (A), raw titin (B), and MyHC (C) values, showing no difference in bandwidth, suggesting similar levels of titin isoforms present in both control and critically ill patients. (D) Migration distance of titin to MyHCII, suggesting little or no difference in titin splicing. (E) Example plot profile, explaining the FWHM and migration measurements. Graph A–D represented as mean ± SD. A reference sample was run on each gel and used to normalize for variations in bandwidth and migration between gels. Bars denote mean ± SEM.