Figure 8
SOCS6-mediated mitochondrial fission is in part mediated by PGAM5 activity. (a–c) HeLa cells stably harboring shRNAs targeting at luciferase (control-KD) and PGAM5 (PGAM5-KD) were established. (a) Total cell lysates were prepared and PGAM5 expression was determined by western blot analysis. (b) Control-KD and PGAM5-KD cells were transiently transfected with pcDNA3 or pcDNA3-HA-SOCS6, and confocal images of MitoTracker and PGAM5 (green, anti-PGAM5) or SOCS6 (green, anti-HA) are shown. Scale bar: 10 μm. (c) Transfected cells in (b) were scored according to their mitochondrial morphology as tubular networks, hyperfused, or fragmented patterns, and the percentages of distribution are shown in the bar graph. A total of 300 cells were counted for each category and data were derived from three independent experiments. (d) AD293 cells were transiently transfected with pcDNA3-HA-SOCS6 alone or with pcDNA3-Flag-PGAM5-L or pcDNA3-Flag-PGAM5-S, and subjected to immunoprecipitation with the anti-Flag beads followed by western blot analysis of the immunoprecipitates using the indicated antibodies. (e) Confocal images of HA-SOCS6 (green, anti-SOCS6) and endogenous PGAM5 (red, anti-PGAM5) in HeLa cells expressing HA-SOCS6. Scale bar: 10 μm. Insets show enlarged views of the boxed areas. (f) Cell lysates were prepared and subjected to immunoprecipitation with the control IgG, anti-DRP1, or anti-PGAM5 beads followed by western blot analysis of the immunoprecipitates using the indicated antibodies
