First level of stringency – extraction with Triton X-100. MBP, tubulin, and actin co-immunoprecipitate from the OLG Triton X-100 insoluble pellet. Primary OLGs were extracted with buffer containing 1% TX-100, and the soluble and resuspended insoluble fractions were immunoprecipitated in the lysis buffer with monoclonal anti-MBP SMI 99 antibody and a control antibody. Western blots were immunostained with rabbit polyclonal anti-MBP (E13) antibody, mouse monoclonal anti-MBP antibody (SMI 99), rabbit polyclonal anti-tubulin antibody, and mouse monoclonal anti-actin antibody. Representative results of 4 experiments are shown. Lane 1, supernatant fraction; lane 2, anti-MBP (SMI 99) immunoprecipitate of supernatant; lane 3, control antibody immunoprecipitate of supernatant; lane 4, pellet; lane 5, anti-MBP (SMI 99) immunoprecipitate of pellet; lane 6, control antibody immunoprecipitate of pellet; lane 7, anti-MBP SMI 99 IgG; lane 8, control antibody IgG. Supernatant fraction was not loaded for the gel used for the actin blot, and the immunoprecipitated supernatant fractions shown in lanes 2 and 3 for the actin blot were from a different gel than the remaining samples, due to overloading and overexposure of the immunoprecipitated supernatant samples on the gel/blot used for the remaining samples. Standards for MBP, tubulin, and actin were not loaded on the gels used for these samples, but can be seen in Figure 2a. The 3 bands for MBP at M
values of approximately 16–27 kDa, indicated by arrows, represent the classic 14-, 18.5-, and 21.5-kDa isoforms of MBP. The 14-kDa isoform was not detected with the monoclonal anti-MBP (SMI 99) antibody but was immunoprecipitated by it, since it was detected by E13 antibody in the anti-MBP (SMI 99) immunoprecipitate (lanes 2,5). Lower-exposure blots show the MBP bands in the immunoprecipitates clearly but did not show those in the pellet or supernatant as well. Therefore, a more highly-exposed blot was chosen for this figure.