Phosphorylation regulates Drp1 and hFis1 binding in vitro. (A) Recombinant Drp1-6His was preincubated in the absence or presence of CaMKIα (KI) and ATP and mixed with GST-hFis1 as indicated. Proteins were recovered using glutathione-Sepharose and analyzed by immunoblotting using an anti-6His antibody to detect Drp1 (top; bottom shows Drp1 input). Quantitation of the results is shown in the bar graph. The results were otained in three independent experiments. Error bars indicate SEM in each group. (B) Drp1-6His was maximally phosphorylated by CaMKI, and the sample was mixed in different ratios with nonphosphorylated Drp1-6His (values are expressed as percent P-Drp1/Drp1; see x axis). GST pull-down assays were performed as in A. The data represent means from three independent experiments. (C) The binding of mutant Drp1-S600A-6His to GST-hFis1 was compared with unphosphorylated and phosphorylated wild-type Drp1-6His. Drp1 was detected by immunoblotting as described above. Similar results were obtained from two independent experiments.