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. 2020 Aug 17;11:4116. doi: 10.1038/s41467-020-17885-z

Fig. 2. OSMR interacts with different components of ETC in human BTSCs.

Fig. 2

a, b Mitochondrial fractions from BTSC73 (a) and BTSC147 (b) were treated with 0.5 mg/mL proteinase K or proteinase K and 1% Triton X-100. Lysates were analyzed by immunoblotting using indicated antibodies. c–f WCL and mitochondrial fractions from BTSC73 (c, d) and BTSC147 (e, f) were subjected to immunoprecipitation using antibodies to OSMR or mouse IgG control, followed by immunoblotting with mtHSP70 and TIM44 antibodies. g, h PLA of OSMR and mtHSP70 were performed in BTSC73 (g) and BTSC147 (h). Primary antibodies were omitted for the controls. i Double labeling of the PLA signal (red) from the OSMR/mtHSP70 interaction and MitoTracker (green) is shown. j OSMR protein expression level was assessed in the mitochondrial fractions obtained from BTSC73 electroporated with siRNA control (siCTL) or siRNA against mtHSP70 (simtHSP70). BLC2 was used as a loading control. k OSMR protein expression level was assessed in the mitochondrial fractions obtained from BTSC73 electroporated with siCTL or siRNA against TIM44 (siTIM44). BCL2 was used as a loading control. l–o WCL or mitochondrial fractions from BTSC73 (l, m) and BTSC147 (n, o) were subjected to immunoprecipitation using an antibody to OSMR or mouse IgG control followed by immunoblotting with NDUFS1 and NDUFS2 antibodies. p, q PLA analyses of OSMR/NDUFS1 and OSMR/NDUFS2 were carried out in BTSC73 (p) and BTSC147 (q). r, s Double labeling of the PLA signal (red) and the MitoTracker (green) is shown. Images were obtained with a 63X objectives on a laser scanning confocal microscope (ZEISS LSM 800). Scale bar = 10 μm. Inset scale bar = 1 μm. Representative images of three independent experiments are shown. The Western blots represent a minimum of three replicates from different passage numbers for each BTSC.