Figure 3

BV treatment reduces the accumulation of ubiquitinated and misfolded SOD1 proteins. After the transient transfection of NSC34 motor neuron cells with GFP or GFP-tagged hSOD1^G85R for 24 hrs, cells were treated with 2.5 μg/ml BV or equal volume of saline for 24 hrs or left untreated. The cells were lysed using RIPA buffer, immunoprecipitated with anti-GFP and then western blot analysis was performed using primary anti-ubiquitin (A) and anti-GFP (B) antibodies. The arrowhead indicates GFP-tagged mutant hSOD1^G85R. Total cell lysates were analyzed by western blotting with anti-GFP (C) and anti-ubiquitin (D) primary antibodies. Asterisks indicate ubiquitinated proteins. To detect misfolded SOD1, NSC34 cells transfected with wild-type or hSOD1^G85R constructs were lysed using the SOD1 polymerization method described previously, and an immunoblot analysis was performed with an anti-misfolded SOD1 antibody (E). The membranes were reprobed with an anti-tubulin antibody as a loading control. G: GFP-transfected cells.