Gapdh ab128915

DIV100-DIV120 astrocytes were grown to 100% confluence and then were manually collected on ice and fresh-frozen cell samples were maintained at −80 °C until further processing. Samples were hydrolyzed in RIPA buffer (containing 50 mM Tris, 150 mM NaCl, 1% (vol/vol) NP40, 0.5% sodium deoxycholate (wt/vol), 0.1% SDS (wt/vol) complemented with protease inhibitors (Complete, Roche Diagnostics, pH 7.6). Protein concentrations were determined using the microBCA kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. In brief, due to limited protein harvest, the entire protein sample was loaded/resolved for all lanes, on a Criterion XT 3–8% Tris-Acetate gel in XT Tricine Running buffer and run as previously described^{13 (link)}. After running, samples were blotted to Nitrocellulose paper using the Trans-blot Turbo Transfer System (Biorad). Blocking was done for 1 h with 5% nonfat milk solution in PBST before incubation with primary antibodies; Dystrophin ab154168 (Abcam, rabbit) 1:2000 Gapdh ab128915 (Abcam, rabbit) 1:10,000. Protein bands were visualized using the Odyssey (Westburg, the Netherlands) after staining with IRDye 680TL (1:2000) and goat-anti-rabbit 926-68021 (Licor-Biosciences) (1:2000) labeled secondary antibodies.

Immunoblotting was performed as previously described [36 (link)]. Cells were lysed with ice-cold RIPA lysis buffer in the presence of a protease inhibitor cocktail. Protein quantification, electrophoresis, and transfer were performed, followed by membrane blocking with 5% nonfat milk. The human-specific antibodies used included GAPDH (ab128915) from Abcam; Tom20 (sc-11415), Parkin (sc-133167), and Tim23 (sc-514463) from Santa Cruz; p62 (23214), PINK1 (6946), and LC3 (3868) from Cell Signaling Technology (Boston, MA, USA); and MFN1 (66776-1-Ig) and MFN2 (67487-1-Ig) from Proteintech (Chicago, IL, USA). Then they were incubated with secondary antibodies (KPL) for 1 h at room temperature. Protein bands were visualized using ECL blotting detection reagents (Life-iLab, Shanghai, China).

Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and propidium iodide (PI) were purchased from Sigma. The following antibodies were used for western blot analysis or immunofluorescence: COX IV (4850), NDP52 (60732), and GST (2625) were purchased from Cell Signaling Technology; monoclonal anti-ubiquitin (sc-8017), anti-Tom20 (sc-17764), anti-PINK1 (sc-517353), and anti-Parkin (sc-32282) were purchased from Santa Cruz; anti-LC3B (L7543) was purchased from Sigma; and anti-Tim23 (611222) was purchased from Becton Dickinson and Company. Actin (EM21002) was purchased from HuaBio. GAPDH (ab128915) was purchased from Abcam.