Figure S2.
CipB has no effect on mitochondrial depolarization and Parkin location. (A) CipB does not alter mitochondrial depolarization in HeLa cells. HeLa cells were transfected with GFP-GFP (yellow), GFP-CipB (yellow), or treated with CCCP (10 μM, 6 h). Mitochondria (cyan) were stained by anti-Tom20 antibody and a ΔΨm-dependent MitoTracker (magenta). Without CCCP treatment, mitochondria are intact and stained by mitochondrial markers. After 6 h of CCCP treatment, mitochondria are depolarized as represented by the loss of MitoTracker staining. Representative images are shown. Scale bar, 40 μm. (B) Quantification of the percentages of cells showing mitochondrial membrane potential in (A). The percentages of cells with mitochondrial membrane potential are means ± SEM; n = 100 cells in each group. (C) CipB does not influence Parkin recruitment to mitochondria. HeLa cells were transfected with the RFP-tagged plasmid (pseudo-colored magenta). As a positive control, cells transfected GFP-Parkin (cyan) were treated with CCCP (10 μM, 6 h), resulting in complete Parkin translocation to mitochondria. Mitochondria (blue) were stained by anti-Tom20 antibody. Scale bar, 10 μm. (D) Quantifications of Parkin-mitochondria colocalization in C. The percentages of Parkin-Tom20 colocalization in GFP-Parkin+ cells were calculated (mean ± SEM, n = 100 cells/group). (E) Schematic view of CipB structure. TMD, transmembrane domain. Numerals indicate the number of aa of CipB. CipB(1–424), aa 1–424 of CipB. The structure of CipB is predicted by TMHMM 2.0 service. (F) Mitochondrial colocalization of CipB variants. HeLa cells transfected with the GFP-tagged plasmids (gray) were analyzed by confocal microscopy. Mitochondria (magenta) were stained by anti-Tom20 antibody. Scale bar, 40 μm. (G) Co-IP analysis of HA-CipB(1-424) with the Flag-tagged mitochondrial protein in 293T cells. Cells were lysed and subjected to Flag IP. The immunoprecipitate was eluted with Flag peptide and analyzed by immunoblotting. (H) Co-IP analysis of TUFM-Flag with the RFP-CipB truncations in 293T cells. Cells were lysed and subjected to Flag IP. The immunoprecipitate was eluted with SDS loading buffer and analyzed by immunoblotting. All data are representative of three independent experiments. Unpaired two-sided Student’s t tests were used to measure significance (B and D). Source data are available for this figure: SourceData FS2.

CipB has no effect on mitochondrial depolarization and Parkin location. (A) CipB does not alter mitochondrial depolarization in HeLa cells. HeLa cells were transfected with GFP-GFP (yellow), GFP-CipB (yellow), or treated with CCCP (10 μM, 6 h). Mitochondria (cyan) were stained by anti-Tom20 antibody and a ΔΨm-dependent MitoTracker (magenta). Without CCCP treatment, mitochondria are intact and stained by mitochondrial markers. After 6 h of CCCP treatment, mitochondria are depolarized as represented by the loss of MitoTracker staining. Representative images are shown. Scale bar, 40 μm. (B) Quantification of the percentages of cells showing mitochondrial membrane potential in (A). The percentages of cells with mitochondrial membrane potential are means ± SEM; n = 100 cells in each group. (C) CipB does not influence Parkin recruitment to mitochondria. HeLa cells were transfected with the RFP-tagged plasmid (pseudo-colored magenta). As a positive control, cells transfected GFP-Parkin (cyan) were treated with CCCP (10 μM, 6 h), resulting in complete Parkin translocation to mitochondria. Mitochondria (blue) were stained by anti-Tom20 antibody. Scale bar, 10 μm. (D) Quantifications of Parkin-mitochondria colocalization in C. The percentages of Parkin-Tom20 colocalization in GFP-Parkin+ cells were calculated (mean ± SEM, n = 100 cells/group). (E) Schematic view of CipB structure. TMD, transmembrane domain. Numerals indicate the number of aa of CipB. CipB(1–424), aa 1–424 of CipB. The structure of CipB is predicted by TMHMM 2.0 service. (F) Mitochondrial colocalization of CipB variants. HeLa cells transfected with the GFP-tagged plasmids (gray) were analyzed by confocal microscopy. Mitochondria (magenta) were stained by anti-Tom20 antibody. Scale bar, 40 μm. (G) Co-IP analysis of HA-CipB(1-424) with the Flag-tagged mitochondrial protein in 293T cells. Cells were lysed and subjected to Flag IP. The immunoprecipitate was eluted with Flag peptide and analyzed by immunoblotting. (H) Co-IP analysis of TUFM-Flag with the RFP-CipB truncations in 293T cells. Cells were lysed and subjected to Flag IP. The immunoprecipitate was eluted with SDS loading buffer and analyzed by immunoblotting. All data are representative of three independent experiments. Unpaired two-sided Student’s t tests were used to measure significance (B and D). Source data are available for this figure: SourceData FS2.

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