![Figure 5. CaMKIα phosphorylates recombinant Drp1 and endogenous Drp1 in cells. (A) Recombinant Drp1 was incubated with either CaMKIα or CaMKII as indicated in the presence of [32P]ATP. Proteins were separated by SDS-PAGE and analyzed by autoradiography and Coomassie staining. (B) Wild-type Drp1 or mutant Drp1 in which Ser600 was replaced by alanine was incubated with CaMKIα and [32P]ATP, and proteins were separated by SDS-PAGE and analyzed by autoradiography. (C) Recombinant Drp1 was incubated with CaMKIα in the absence or presence of ATP. Proteins were separated by SDS-PAGE and analyzed by immunoblotting using a phosphospecific antibody selective for phospho-Ser600. (D) HeLa cells were transfected without (control siRNA) or with siRNA selective for CaMKIα. HeLa cells were incubated without (−) or with 45 mM K+ for 15 min (+). Proteins were separated by SDS-PAGE and analyzed by immunoblotting with phospho-Ser600 or Drp1 antibodies. (E) Neurons were incubated for the indicated times with 45 mM K+ in the absence or presence of KN93 (20 μM added 30 min before high K+). Proteins were separated by SDS-PAGE and analyzed by immunoblotting with phospho-Ser600 or Drp1 antibodies as indicated. (F) Neurons were untreated (control) or treated with 45 mM K+ for 15 min. Proteins were separated by SDS-PAGE and analyzed by immunoblotting with phospho-Ser600 or total Drp1 antibodies as indicated (bottom). Bar graphs show cumulative data from three independent experiments for each condition. Error bars indicate SEM in each group. *, P < 0.05.](https://cdn.rupress.org/rup/content_public/journal/jcb/182/3/10.1083_jcb.200802164/6/jcb_200802164_gs_fig5.jpeg?Expires=1767780143&Signature=MFtSMotguTzOk0BD98ZlAeub9RVdFYuYroXh7kEl4rY-jwOFT~5YkaR5N22Nj5dcLuxAP4hyTKh8dX9B3lknadsuc7XYygR4bCc1Dv6s~dGhFhihzRVM~MjIeQRijD0rVBLd4Tivg8x44VHB-mkWP5bgKH4eWvjlybkc~xs3m7Xw3ALWk5XHAWiyr4mfWGKBZTUtCW7mA5w1BdIsHPcVG4v2Fpbs8b68YgA-POiFliCatU8rZZ9K261CklO2~WtMFlFca3iEi7OlMXNqvkzO4C2chuq4lY0EC-Xqu1lJPVTRltsCr~R27MCIDm2bBLesDUrwCj8C-uILotvLlPbtoQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CaMKIα phosphorylates recombinant Drp1 and endogenous Drp1 in cells. (A) Recombinant Drp1 was incubated with either CaMKIα or CaMKII as indicated in the presence of [32P]ATP. Proteins were separated by SDS-PAGE and analyzed by autoradiography and Coomassie staining. (B) Wild-type Drp1 or mutant Drp1 in which Ser600 was replaced by alanine was incubated with CaMKIα and [32P]ATP, and proteins were separated by SDS-PAGE and analyzed by autoradiography. (C) Recombinant Drp1 was incubated with CaMKIα in the absence or presence of ATP. Proteins were separated by SDS-PAGE and analyzed by immunoblotting using a phosphospecific antibody selective for phospho-Ser600. (D) HeLa cells were transfected without (control siRNA) or with siRNA selective for CaMKIα. HeLa cells were incubated without (−) or with 45 mM K+ for 15 min (+). Proteins were separated by SDS-PAGE and analyzed by immunoblotting with phospho-Ser600 or Drp1 antibodies. (E) Neurons were incubated for the indicated times with 45 mM K+ in the absence or presence of KN93 (20 μM added 30 min before high K+). Proteins were separated by SDS-PAGE and analyzed by immunoblotting with phospho-Ser600 or Drp1 antibodies as indicated. (F) Neurons were untreated (control) or treated with 45 mM K+ for 15 min. Proteins were separated by SDS-PAGE and analyzed by immunoblotting with phospho-Ser600 or total Drp1 antibodies as indicated (bottom). Bar graphs show cumulative data from three independent experiments for each condition. Error bars indicate SEM in each group. *, P < 0.05.
