Second level of stringency – extraction with Triton X-100 and NP40 – cortactin blot. MBP and cortactin co-immunoprecipitate from the OLG Triton X-100 plus NP40-insoluble pellet and supernatant fractions. Lane 1, supernatant fraction; lane 2, blank; lane 3, pellet; lane 4, anti-MBP (SMI 99) immunoprecipitate of supernatant; lane 5, control antibody immunoprecipitate of supernatant; lane 6, blank; lane 7, anti-MBP (SMI 99) immunoprecipitate of pellet; lane 8, control antibody immunoprecipitate of pellet; lane 9, anti-MBP SMI 99 IgG; lane 10, control antibody IgG. The supernatant and pellet fractions were run on a different gel from the immunoprecipitated samples. The blots were stained with rabbit polyclonal anti-cortactin antibody. Two bands at about 78 kDa and 82 kDa were detected for cortactin. Representative results are shown from 2 experiments.