Bp1408 1

Cells were scraped in homogenization-A buffer, sonicated, and protein concentration was measured using the Bradford Plus Assay Reagent (ThermoFisher, PI23236). Polyacrylamide gels (10% 29:1 polyacrylamide/bisacrylamide (Fisher BioReagents, BP1408-1), 375 mM Tris pH 8.8 (Fisher BioReagents, BP152-1), 0.1% sodium dodecyl sulfate (SDS, Sigma, L3771), 0.1% ammonium persulfate (APS, Sigma, #A3678), and 0.1% TEMED (GE Healthcare, # 45-000-226)) were loaded with 10 μg of whole cell lysates and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies (1:1000 Mouse Anti-Dlp1/Drp1 in 2% milk (Clone 8, BD Transduction Labroraotries, DB6111112); 1:1000 Mouse Anti-Opa1 in 2% BSA (DB Transduction Laboratories, DB612607); 1:10,000 Rabbit Anti-GAPDH in 2% BSA (14C10, Cell Signaling, 2118)) at 4 °C overnight. Membranes were then washed with Tris-Buffered Saline and 0.1% Tween (TBST, Fisher Scientific, BP337500) and incubated in secondary antibodies for 60 min at room temperature. Membranes were then washed 3 × with TBST for 5 min. The membranes were incubated in SuperSignal West Pico Plus Chemiluminescent Substrate (ThermoFisher, 34577), imaged utilizing a BioRad ChemiDoc XRS and imager, and finally quantified by densitometry using ImageJ.

Cells were scraped in homogenization-A buffer (with MG132 proteasome inhibitor when probing for ubiquitination), sonicated, and the protein concentration was measured using the Bradford Plus Assay Reagent (ThermoFisher, PI23236). Polyacrylamide gels (10% 29:1 polyacrylamide/bisacrylamide (Fisher BioReagents, BP1408-1), 375 mM Tris pH 8.8 (Fisher BioReagents, BP152-1), 0.1% sodium dodecyl sulfate (Sigma, L3771), 0.1% ammonium persulfate (APS, Sigma, #A3678), and 0.1% TEMED (GE Healthcare, #45-000-226)) were loaded with 10 μg of whole cell lysates and 5 μg of mitochondrial samples and transferred to nitrocellulose membranes. Membranes were incubated with indicated primary antibodies at 4 °C overnight. Membranes were then washed with Tris-buffered saline and 0.1% Tween (TBST, Fisher Scientific, BP337500) and incubated in secondary antibodies for 60 min at room temperature. Membranes were washed 3× with TBST, for 5 min. The membranes were then incubated in SuperSignal West Pico Plus Chemiluminescent Substrate (ThermoFisher, 34577), imaged utilizing a BioRad ChemiDoc XRS+ imager and quantified by densitometry using ImageJ software.