Figure 4.

RalBP1 promotes phosphorylation of Drp1. A Immunoblot analysis of S616 phosphorylated (p-S616-Drp1), Drp1, RalA, RalBP1 and actin levels in HeLa cells expressing a scramble control, RalA shRNA or RalBP1 shRNA. Graph represents mean p-S616-Drp1 signal intensity measured by imageJ from 3 experiments +/− SD (** p = 0.008). B Extracts isolated from HeLa cells, either unsynchronized (U) or synchronized in mitosis (M) using double thymidine block were either analyzed by immunoblot for levels of RalA, Drp1, cyclin B or S616 phosphorylated Drp1, or subjected to immunoprecipitation (IP) with either no antibody or antibodies against cyclin B or RalBP1, followed by incubation with GST-Drp1 and γ³²P-ATP, and resolved on an acrylamide gel. ³²P-labeled GST-Drp1 was visualized by phosphorimager while total GST-Drp1 was visualized by coomassie brilliant blue staining. Graphs represent mean ³²P-GST-Drp1 signal intensity measured by imageJ from 3 experiments +/− SD (* p = 0.028 for Cyclin B, 0.027 for RalBP1). C 200ng GST-Drp1 and 25ng GST-Cyclin B/Cdk1 were incubated with increasing amounts of either GST-RalBP1 or GST (0, 125, 250, 500, 1000ng) in addition to either ATP or γ³²P-ATP. Reactions were resolved by SDS-PAGE and either subjected to immunoblot for Drp1, RalBP1 and S585 phosphorylated Drp1 (cold kinase assay) or visualized by phosphorimager (hot kinase assay). GST, GST-Drp1 and GST-RalBP1 were visualized by coomassie brilliant blue staining (CBB). Graph represents mean p-S616-Drp1 signal intensity measured by imageJ from 3 experiments +/− SD (* p = 0.020, 0.028 ** p = 0.009). D Immunoblot analysis of RalA, RalBP1, Drp1 and Cyclin B levels in crude mitochondrial fractions (mito) and whole cell extracts (WCE) isolated from HeLa cells synchronized in mitosis (M) using double thymidine block, treated with DSP, and subjected to immunoprecipitation with and anti-RalA antibody.