P parkin

Cells were rinsed with cold PBS and harvested in lysis buffer [31] (link). Then, the extractions were obtained and then centrifuged at 14,000 rpm for 30 min. Twenty-five micrograms of protein was loaded per lane and separated by SDS-PAGE, then transferred to nitrocellulose membranes and blocked overnight in 5% skim milk. Then, the membrane was incubated with primary antibodies at 4 °C and subsequently incubated with a secondary antibody for 2 h at room temperature. The primary antibodies for the blots are as follows: p-CaMKII (1:1000, Cell Signaling Technology, Inc.), p-cofilin (1:1000, Abcam plc), CaMKII (1:1000, Cell Signaling Technology, Inc.), cofilin (1:1000, Cell Signaling Technology, Inc.), Beclin1 (1:1000, Cell Signaling Technology, Inc.), LC3II (1:1000, Cell Signaling Technology, Inc.), ATG5 (1:1000, 1:1000, Abcam plc), Yap (1:1000, Cell Signaling Technology, Inc.), F-actin (1:1000, 1:1000, Abcam plc), G-actin (1:1000, 1:1000, Abcam plc), p-JNK (1:1000, Cell Signaling Technology, Inc.), JNK (1:1000, Cell Signaling Technology, Inc.), Parkin (1:1000, Cell Signaling Technology, Inc.), p-Parkin (1:1000, Cell Signaling Technology, Inc.), Bnip3 (1:1000, Cell Signaling Technology, Inc.), p62 (1:1000, 1:1000, Abcam plc) and SERCA (1:1000, Cell Signaling Technology, Inc.)

Western blotting was performed as previously described [42 (link)]. The primary antibody for the following protein were used: GAPDH (Cell signaling technology, #51332), β-actin (Abcam, #ab8227), Shank3 (Abcam, #ab264347), Nox4 (Abcam, #ab13303), cleaved-capase9 (Abcam, #ab184786), PINK1(Abcam, #ab23707), Parkin (Abcam, #ab77924), p-Parkin (Cell signaling technology, #36866), LC3 (Cell signaling technology, #4108), P62 (Abcam, #ab91526), Beclin1 (Abcam, #ab62557), Bax (Abcam, #ab53154), Bcl-2 (Abcam, #ab59348), CaMKII (Cell signaling technology, #3362), P16 (Abcam, #ab151303), P21(Abcam, #ab109199), P53 (Abcam, #ab131442), PGC1-α (Abcam, #ab54481), TFAM (Cell signaling technology, #7495), NRF-1 (Abcam, #ab55744).
To detect protein levels in the mitochondrial fraction, cytosol, and total cell fractions, a mitochondria/cytosol kit (Beyotime, China) was used to isolate mitochondria and cytosol, according to the manufacturer’s protocol. Each fraction was quantified using a protein quantitation kit. For western-blot analysis, each fraction was loaded based on equal protein quantity (30 μg) and Western blot analysis was performed. GAPDH served as a loading control for the total cell and cytosol fractions, whereas VDAC1 was used as a loading control for the mitochondrial fraction.