Affinipure goat anti mouse igg and goat anti rabbit igg

Cells were lysed in ice-cold 2X laemmli sample buffer (0.125 M Tris-HCl, 20% glycerol, 4% sodium dodecyl sulfate and 0.004% Bromophenol blue) with additional protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors cocktail (Roche). The total protein extracts were separated by SDS-PAGE and transferred to a 0.2 μm nitrocellulose membrane. Membrane was probed with primary antibody for 1 hour at room temperature or overnight incubation at 4°C followed by incubation in peroxidase-conjugated AffiniPure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) at 1:2000 dilution. To detect proteins of interest, peroxidase substrate was applied for enhanced chemiluminescence.

Western blotting was conducted as in the previous studies [19 (link), 20 (link)]. Primary antibodies used herein include those targeting ND6 (Abcam, ab81212), ND4 (Abcam, ab219822), ATP6 (Proteintech, 55313-1-AP), ND1 (Abcam, ab74257), CYB (Proteintech, 55090-1AP), CO2 (Abcam, ab110258), and VDAC (Proteintech, 10866-1-AP). As a secondary detection antibody, we employed the peroxidase AffiniPure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) constructs, and an enhanced chemiluminescence (ECL) system (Millipore) was used for band detection, after which densitometric calculations were performed.