Anti atpb

Cells were lysed at 4 °C in extraction buffer (0.1 M NaCl, 20 mM HEPES pH 7.5, 1 mM EDTA, 5 mM NaF, 1 mM dithiothreitol, 0.3% Triton X-100, 5% glycerol, 0.25 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor cocktail and phosphatase inhibitors). Homogenates were cleared by centrifugation (12,000 g, 10 min). Coimmunoprecipitation was performed according to previous techniques^{55 (link)}. In vitro IP was performed using purified recombinant proteins instead of the cell lysate. The antibodies used in the study were as follows: Anti-Myc (Cell Signaling, #2276); Anti-DDK (Origene, TA50011); Anti-DJ-1(Santa Cruz, sc27006 and sc32874; Cell Signaling, 2134S); Anti-β-Actin, #4970); Anti-GAPDH (Santa Cruz, sc-32233); Anti-mATP5G1/2/3(abcam, ab180149); Anti-ATPB (abcam, ab14730), and Anti-Bcl-xL(Cell Signaling, #2764); Anti-Puromycin (3RH11) (Kerafast, EQ0001).

BMDMs were washed twice, fixed by 4% cold paraformaldehyde for 30 min, permeabilized by 0.5% Triton X‐100 for 10 min, blocked by goat serum for 1 h, and incubated with anti‐ATPB (1:100, Abcam) overnight at 4°C. On the second day, BMDMs were stained with Goat anti‐mouse IgG H&L (Alexa Flour® 488, Abcam) for 1 h, followed by a second block and primary incubation (anti‐dynamic‐related protein 1 (anti‐Drp1), 1:250, Abcam; anti‐SQSTM1/p62, 1 μg/mL, Abcam). On the third day, a secondary antibody (Alexa Flour® 555, Abcam) was added to incubate with cells for 1 h. DAPI was used to stain the nucleus. Slides and stained cells were observed and photographed by a confocal microscope.