Ab 54481

Proteins from tissues or cultured cells were extracted, transferred onto
PVD-membranes, hybridized overnight with appropriate primary antibodies
(PPARGC1A - sc-13067 and ab-54481, DIO2-sc-98716, BCL-xL- ab32124,
PINK1-ab23707, LC3B-ab51520, p62/SQSTM1-ab56416, BAX-ab32503 and ab-53154,
β-actin-sc-47778) obtained from Santa Cruz Biotechnology, CA (sc), or
Abcam, Cambridge, UK (ab) and visualized using the Gel Doc XR+ System
(Biorad, Lab. Inc. Life Science, USA), according to manufacturer’s
protocol.

Cells were lysed in RIPA buffer (10 mM Tris-HCL, 1 mM EDTA, 1% sodium dodecyl sulfate [SDS], 1% Nonidet P-40, 1: 100 proteinase inhibitor cocktail, 50 mM β-glycerophosphate, 50 mM sodium fluoride). The samples were separated on a 10% SDS-PAGE gel and transferred to PVDF membranes by a semi-dry transfer apparatus (Bio-Rad). After blotting with 5% non-fat milk for 1 hour, the membranes were incubated with primary antibodies at 4˚C overnight (Abcam Cat#ab54481,1:1000 for anti-PGC-1α; Santa Cruz Cat#sc21382, 1:1000 for anti-NRF2; Novus Biologicals Cat#NB110–58359, 1:1000 for anti-TAZ; Sigma-Aldrich, Cat#T9026, 1:10000 for α-tubulin). The immunocomplexes were incubated with horseradish peroxidase-conjugated anti-rabbit or antimouse IgG (Promega) and detected with SuperSignal reagents (Pierce) as previously described (Chang et al., 2009 (link)). For
Cell Stem Cell 23, 193–209.e1–e5, August 2, 2018 e4 quantification of western blots, band intensities for each antibody-specific blots were measured as areas under the intensity profile curve, using the ImageJ software. Relative band intensity for each lane was calculated after normalization against intensity for α-tubulin in the corresponding lane.