Gastrocnemius Muscle Protein Analysis

WB analysis of gastrocnemius muscles was performed as previously reported [21 (link)]. If mitochondrial protein isolation is required, it is done using the mitochondrial protein extraction kit (BB-3171, Best Bio, Shanghai, China). Primary antibodies include Stat3 (1 : 1000), P-Stat3 (1 : 1000), HIF-1α (1 : 1000), BNIP3 (1 : 5000), Beclin 1 (1 : 1000), Atg7 (1 : 1000), LC3A/B (1 : 1000), p62 (1 : 1000), GAPDH (1 : 2500), and COX IV (1 : 2000). After the primary antibody protocol was completed, the corresponding secondary antibody (1 : 5000) was added according to the protocol. The density values of the bands were captured and documented through a gel image analysis system (ChemiDox™, Bio-Rad, USA) and normalized to GAPDH or COX IV; n = 3.