Figure 4

Third level of stringency – extraction with Triton X-100 and NP40 and DOC. MBP, tubulin, actin, and ZO-1 do not co-immunoprecipitate in 1% TX-100 plus 1% NP40 plus 1% DOC; addition of DOC disrupts the protein complexes. Primary OLGs were extracted with buffer containing 1% TX-100 plus 1% NP40 plus 1% DOC, the soluble and resuspended insoluble fractions were immunoprecipitated with mouse monoclonal antibody anti-MBP SMI 99 and a control antibody. Western blots were immunostained with rabbit polyclonal anti-MBP (E13), rabbit polyclonal anti-tubulin, mouse monoclonal anti-actin, and rat monoclonal anti-ZO-1 antibodies. Lane 1, standard purified protein sample (18.5-kDa isoform for MBP), except for actin and ZO-1; lane 2, supernatant fraction; lane 3, anti-MBP (SMI 99) immunoprecipitate of supernatant; lane 4, control antibody immunoprecipitate of supernatant; lane 5, pellet; lane 6, anti-MBP (SMI 99) immunoprecipitate of pellet; lane 7, control antibody immunoprecipitate of pellet; lane 8, anti-MBP SMI 99 IgG; lane 9, control antibody IgG.