Figure 5
Gykl1/Gk2 interacts with Pld6 and stimulate mitochondrial clustering. (a) HEK293T cells transiently co-expressing FLAG-tagged Pld6 with GST-tagged Gyk, Gykl1 or Gk2 were immunoprecipitated (IP) with anti-FLAG antibodies. The immunoprecipitates were blotted with antibodies against GST or FLAG. An antibody against GAPDH was used for loading control. (b) Bacterially-purified GST-tagged recombinant Pld6 lacking the mitochondria localization sequence (GST-Pld6ΔMLS), was mixed with purified MBP-tagged recombinant Gykl1 lacking the C-terminal 29 amino acids (MBP-Gykl1ΔC29) for in vitro GST pull-down assays. The reaction mixtures were resolved by SDS-PAGE and stained with Coomassie blue. Recombinant GST alone served as a negative control. (c) HEK293T cells transiently co-expressing GFP-PABD with HA-tagged Pa-pla1, Pld6, Gyk, Gykl1 or Gk2 were stained with an anti-HA antibody and the MitoTracker dye. Hoechst 33342 was used to visualize the nuclei. PABD is the Raf1 Phosphatidic Acid Binding Domain. Arrows indicate areas of mitochondrial clustering. Scale bar, 10 μm. (d) HEK293T cells transiently co-expressing GFP-Gykl1 with HA-tagged mCherry, Pld6 or Pa-pla1 were immunostained with an anti-HA antibody. Hoechst 33342 was used to visualize the nuclei. Scale bar, 10 μm. (e) Cells from d were scored for cells with obvious mitochondrial clustering in three independent experiments (n=3) and the percentages of cells with mitochondrial clustering are plotted here. At least 200 cells were counted for each group. Error bars represent s.d. Statistical significance was determined by ANOVA; *P<0.05. (f and g) HEK293T cells co-expressing GFP-Gk2 with HA-tagged mCherry, Pld6, or Pa-pla1 were similarly examined (f) and analyzed (g) as described in d and e. At least three independent experiments were performed with >200 cells counted in each group. *P<0.05. Scale bar, 10 μm.
