Figure 3. CRMP2 is minimally SUMOylated by multiple SUMO isoforms. CAD cells were transfected with Flag-CRMP2 (A) or putative SUMOylation conjugation site mutant Flag-CRMP2AAA (B) and 2 μg pCDNA3 Ubc9 alone or in the presence of pCDNA3 HA-SUMO1, pCDNA HA-SUMO2, or pCDNA HA-SUMO3. HA-tagged (top blots) proteins were purified and immunoblotted with an anti-Flag antibody. Lysates, representing < 1% of the input used for the immunoprecipitations, were immunoblotted with an anti-Flag antibody to reveal CRMP2. SUMOylated CRMP2 was detectable in the presence or absence of Ubc9; this represents less than < 1% of the total cellular CRMP2. Representative blots from three independent experiments are shown. Following incubation with enhanced ECL, the top blot in (A) was exposed for over an hour to film to resolve the really low signal.