MT-targeted Drp1 is a major isoform at mRNA and protein levels. (A) RNA from the indicated mouse tissues was analyzed by qRT-PCR with Drp1 splice-specific primer sets (means ± SEM, n = 3). (B) Triton X-100 extracts of the indicated mouse tissues (15–30 µg/lane) were subjected to immunoblotting for the indicated proteins, and relative expression of Drp1-x01 was quantified as the ratio of x01 to pan-Drp1 antibody signals standardized by signals in an adjacent lane with extract containing GFP-Drp1-001 (100%; means ± SEM, n = 3). (C–E) Quantification of Drp1 isoform expression and phosphorylation by 2D immunoblotting is shown. (C) Total PC12 lysates were treated with or without alkaline phosphatase and separated by 2D PAGE. One set of blots was coincubated with antibodies to total and Ser_CDK-phosphorylated Drp1, whereas a second set was probed for Drp1-x01. (D) Similar 2D immunoblotting experiments identify Drp1 splice and phosphorylation variants in Jurkat and HeLa cells. Trapezoids highlight the major Drp1 splice variant constellation ± Ser_CDK phosphorylation; dashed circles indicate a hyperphosphorylated form of Drp1-001 generated by PP1/PP2A inhibition via calyculin A (25 nM, 1 h). (E) Relative Drp1 splice variant protein expression was quantified by densitometry of 2D blots probed for total Drp1 (means ± SEM, n = 3).