Enrichment and Immunoprecipitation of Cellular Proteins

Whole cell lysates were prepared by lysing cells with a buffer containing 20 mM Tris, pH 7.4, 137 mM NaCl, 25 mM β-glycerophosphate, 2 mM sodium pyrophosphate, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 10% glycerol, and 1 × protease inhibitor cocktail (Roche). Whole cell lysates were incubated with GFP-Trap-A beads or RFP-Trap Agarose beads (ChromoTek) to enrich and isolate Y/RFP or Y/RFP-tagged proteins as described previously (Guo et al., 2017 (link)). Whole cell lysates were incubated with SUMO-2/3 affinity agarose beads (ASM24; Cytoskeleton) at 4°C for overnight to enrich and isolate SUMO-2/3 or SUMO-2/3 conjugates. Whole cell lysates were incubated with glutathione-sepharose 4B beads (Generon) to enrich and isolate GST or GST-tagged proteins as described previously (Guo et al., 2017 (link)). To immunoprecipate endogenous Mff, Bcl-x_L or Drp1, whole cell lysates were incubated overnight with an Mff rabbit polyclonal antibody (17090-1-AP, Proteintech), a Bcl-x_L rabbit monoclonal antibody (54H6, Cell Signaling) or a Drp1 rabbit monoclonal antibody (D6C7, Cell Signaling), pre-conjugated to protein-A beads (Sigma), respectively. The protein-A beads were spun down, washed three times and resuspended in SDS sample buffer for immunoblot analysis.