Ndufs2

Here, 20 µg of total cellular proteins obtained from MSCs were denatured and loaded on sodium dodecyl sulfate polyacrylamide gels. After electrophoresis, the gels were transferred to a PVDF membrane (Millipore) and processed for immunoblotting. Commercially available antibodies, such as MTU1 (ab50895), ND5 (ab92624), NDUFB8 (ab110242), SDHB (ab14714), UQCRC2 (ab14745), ATP5a (ab14748), and MTCO1 (ab17405) from Abcam, ATP6 (55313-1-AP), GAPDH (60004-1-Ig), and VDAC (55259-1-AP) from Proteintech, NDUFS1 (A16926), NDUFS2 (A12858), ND4 (A17970), and CYTB (A17966) from Abclonal were used. Peroxidase AffiniPure Goat Anti-Mouse IgG and Goat Anti-Rabbit IgG (Jackson) were used as secondary antibodies, and the protein signals were detected using the ECL system. Band intensities were quantified from the 16-bit digital image by densitometry in ImageJ and are shown normalized to GAPDH for each target.

For western blot analysis, mitochondria or cell pellets were lysed in RIPA buffer (50 mm Tris‐HCl, 150 mm NaCl, 0.2% SDS, 0.5% sodium deoxycholate, 1% Triton X‐100, 5% glycerol) containing freshly added protease inhibitors (Thermo Fisher), 2 mm Na₃VO₄, 1 mm DTT and 1 mm sodium‐β‐glycerophosphate, and incubated on ice for 10 min. Lysates were spun at 20 000 g for 10 min, and clarified lysates were resolved on SDS/PAGE, transferred to Polyvinylidene fluoride membranes, blocked with 5% milk, and probed with the antibodies. Unless otherwise specified, all antibodies and detection reagents were used at 1 : 2000 dilution prepared in 5% BSA in TBST: STAT3‐alpha (D1A5) (CST, Danvers, MA, USA, catalog no. 8768S), STAT3 (124H6) (CST, catalog no. 9139S), NDUFV2 (ABclonal, Woburn, MA, USA, catalog no. A7442), NDUFS2 (ABclonal, catalog no. A12858), NDUFAF2 (ABclonal, catalog no. A14296), SDHA (ABclonal, catalog no. A2594), Tubulin (Sigma, catalog no. 26628228), Streptavidin‐HRP (BD, Franklin Lakes, NJ, USA, catalog no. 554066). The blots were developed using a chemiluminescent detection kit (Advansta WesternBright ECL HRP substrate) on a LI‐COR Odyssey® Fc Imaging System.