Mitochondrial protein extraction kit

Mitochondria were isolated by mechanical means and differential centrifugation [64 (link)] in accordance to the manufacturer's instructions in a mitochondrial protein extraction kit (Bestbio, China). Briefly, a total of 2 × 10⁷ cells were harvested by centrifugation at 500×g for 5 min and washed 3 times with 1×PBS. The cells were resuspended with Solution A and homogenized on ice with a glass homogenizer. Following that, a low-speed spin (1,000×g) at 4°C for 5 min pelleted unbroken cells, membranes, and nuclei. The supernatant underwent a subsequent high-speed centrifugation (15,000×g, 20 min) to sediment mitochondria, which were then resuspended in Solution B and subjected to the same high-speed spin. Protein was extracted from the crude mitochondria, which were disrupted in protein lysis buffer with protease inhibitors cocktail [4-(2-aminoethyl) benzenesulfonyl fluoride, aprotinin, E-64, leupeptin, bestatin, EDTA and pepstatin A], and kept at −80°C until used.

Mitochondrial membrane proteins were extracted using a mitochondrial protein extraction kit (Bestbio, Shanghai, China). The protein concentrations in the supernatants were determined using the BCA protein assay (Bestbio, Shanghai, China) according to the manufacturer’s instructions. To analyze CPT1 activity, a reaction mixture containing Tris-buffer (100 mM, pH 8.0, 0.1% Triton X-100, 1 mM EDTA), 0.01 mM palmitoyl CoA, and 0.5 mM DTNB was added to each sample, and then the absorbance at 405 nm was measured using a microplate reader. Thereafter, L-carnitine (1.25 mM) was added, incubated for 30 min, and the absorbance at 405 nm was measured.

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.