Figure 2.

Drp1 is modified by SUMO1, SUMO2, and SUMO3. A) HEK-293 cells were transfected transiently with expression vectors for pcDNA3-Ubc9 and/or pcDNA3-HA-SUMO1, pcDNA3-HA-SUMO2, or pcDNA3-HA-SUMO3 and/or pcDNA3-V5-His-Drp1. Cells were lysed in the presence of 20 mM NEM, and His-Drp1 was isolated via Ni²⁺ chelate chromatography under denaturing conditions. Proteins from Ni²⁺ eluates and extracts were visualized by Western immunoblot (IB) analysis using antibodies against Drp1, HA-epitope, and GAPDH. Migrations of unmodified and SUMO-conjugated forms of Drp1 are indicated by asterisk and arrowheads, respectively. Open circle indicates larger apparent molecular mass Drp1 forms likely representing entangled SDS-resistant oligomers. B) SUMO modification of Drp1 is Ubc9 and SUMO3 dependent and NEM sensitive. HEK-293 cells transiently transfected with pcDNA3-Ubc9 and pcDNA3-HA-SUMO3 and/or pcDNA3-V5-His-Drp1 were lysed in the presence or absence of 20 mM NEM and analyzed as in A. C) Cells were cotransfected with HA-Drp1 and Myc-SUMO1, or else transfected with Myc-SUMO1 alone, and cell lysates were immunoprecipitated using anti-HA antibodies as indicated (α-HA IP), then immunoblotted using anti-Myc antibodies. Arrowheads indicate SUMO-modified Drp1 forms. Myc-Drp1 expression (asterisk) identifies the size of unmodified Drp1, as it is identical in size to HA-Drp1. D) Cells were cotransfected with HA-Drp1 (aa 326–736) and Myc-SUMO1, and extracts were immunoprecipitated with anti-HA antibodies and then immunoblotted for Myc as in C. Arrowheads indicate SUMO-modified Drp1 (aa 326–736) forms. Myc-Drp1 (aa 326–736) expression (asterisk) identifies the size of unmodified Drp1, which is identical in size to HA-Drp1 (aa 326–736). Migrations of molecular mass standards (kDa) are at left.