Second level of stringency – extraction with Triton X-100 and NP40. MBP, tubulin, actin, ZO-1, and Fyn co-immunoprecipitate from the OLG TX-100 plus NP40-insoluble pellet and supernatant fractions. Primary OLGs were extracted with buffer containing 1% TX-100 plus 1% NP40, and the supernatant and resuspended pellet were immunoprecipitated with mouse monoclonal antibody anti-MBP SMI 99 and a control antibody. Western blots were immunostained with (a) rabbit polyclonal anti-MBP (E13), rabbit polyclonal anti-tubulin, mouse monoclonal anti-actin, rat monoclonal anti-ZO-1, and (b) mouse monoclonal anti-Fyn. Representative results are shown from 3 experiments for MBP, tubulin, and actin, and from 2 experiments for ZO-1 and Fyn. (a, b) lane 1, standard purified protein sample (18.5-kDa isoform for MBP), except for ZO-1 and Fyn (the tubulin sample was run on the same gel as the other lanes, but separated from them by a M
marker lane and, therefore, was cut out and shown separately to preserve lane alignment); 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. Arrows for the MBP blot indicate the 21.5-, 18.5-, 17-, and 14-kDa isoforms of MBP. Lower-exposure blots showed the MBP bands in the immunoprecipitates clearly, but less well for the pellet or supernatant. Therefore, a more highly-exposed blot was chosen for this figure. The M
of ZO-1 is about 220 kDa, but this antibody also detects several unidentified bands down to 110 kDa in rat liver, according to the manufacturer. (b) Fyn was detected in the pellet and supernatant at M
of about 60 kDa (lanes 2,5).