Western blot was completed as previously described by us [5 (link),21 (link),22 (link)]. In brief, the soleus muscle was homogenized using radioimmunoprecipictation assay cell lysis buffer and the supernatant was used. Using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA), protein concentration was estimated and 50 µg of protein was run onto a 10% or 15% SDS-PAGE gel (150 V for 1 h). Using a semi-dry transfer machine (Bio-Rad), gels were transblotted onto polyvinylidene difluoride membranes (Bio-Rad) and were subsequently blocked with 5% skim milk. Next, the membranes were incubates with primary antibodies overnight at 4 °C for inflammasome markers including TLR4 (1:1000, Cat# ab13556, 90 kDa; Abcam) and NLRP3 (1:1000, Cat# ab214185, 90 kDa; Abcam). Pyroptosis markers were detected using primary antibodies for IL-1β (1:1000, Cat# ab2105, 31 kDa; Abcam), and IL-18 (1:1000, Cat# ab7149, 22 kDa; Abcam). Inflammation was detected by primary antibody for TNF-α at a 1:500 dilution (Cat# ab6671, 17–22 kDa; Abcam). Finally, primary antibody for β-actin was used as a loading control at a 1:1000 dilution (Cat# ab16039, 45 kDa; Cell Signaling Technology, Danvers, MA, USA). Membranes were exposed to enhanced chemiluminescence substrate (ThermoFisher Scientific). Densitometric analysis was performed using NIH ImageJ software.
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