Fig. 3. Silencing of SENP5 results in increased DRP1 SUMOylation. (A) SENP5 silencing increased DRP1 conjugates. COS-7 cells transfected with either shRNA vector alone (vector) or with SENP5-specific shRNA (SENP5 RNAi1), were selected in the presence of 5ug/ml puromycin for 12 days before collection; 100 µg of the total lysates were separated by SDS-PAGE and western blots with the indicated antibodies are shown. (B) Quantification of the SENP5 and DRP1 protein levels in control (Vector) or SENP5-silenced [shRNA (SENP5)] cells from eight different experiments. In each case, 100 µg of total cell extract was loaded and signals from the western blots were quantified using the Alpha Innotech HD Gel documentation system. (C) Immunoprecipitation of DRP1 from control (Vector) or SENP5-silenced (RNAi1) cells reveals an increase in mono-SUMOylated DRP1. COS-7 lysates from cells stably expressing vector alone or SENP5 shRNA were immunoprecipitated with either anti-DRP1 antibody or mouse IgG. Immunoprecipitates were run in duplicates on a 4-20% gradient gel; one blot was probed for endogenous DRP1 and the other for SUMO1. (D) Immunoprecipitation of DRP1 from SUMO1:FLAG-transfected COS-7 cells stably expressing vector alone or SENP5 shRNA. COS-7 cells stably expressing vector alone or SENP5 shRNA were transfected with SUMO:Flag for 24 hours prior to immunoprecipitation with either anti-DRP1 antibody or mouse IgG. Immunoprecipitates were run on duplicate gels; one blot was probed with a monoclonal anti-Flag antibody, the second with the anti-SUMO antibody. Following this, both gels were probed with anti-DRP1 antibody. The increased total level of DRP1 precipitated within the SENP5-silenced cells appears as a doublet. Only the top band of this doublet overlaps directly with the anti-Flag and anti-SUMO1 antibody signal (arrow).