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Fig. 2. Cortactin is required for the formation of clathrin-coated vesicles. (A) Rat brain extracts were depleted with beads conjugated with cortactin antibody as described in Materials and Methods. Depletion of cortactin was verified by cortactin immunoblot analysis. Non-treated brain extracts at different amounts were also analyzed (lane 1, 12 µl; lane 2, 6 µl; lane 3, 3 µl). Lane 4, 12 µl of cortactin depleted extracts; lane 5, cortactin antibody conjugated beads that had been used to absorb brain extracts; lane 6, unused cortactin antibody beads. (B) 3T3-L1 cells were permeabilized by freezing and thawing. The permeabilized cells were incubated with B-SS-Tfn at 4°C for 20 minutes and then mixed with mock-depleted brain extracts (Mock), recombinant cortactin protein in the same buffer as extracts plus 1% BSA (Cort only), cortactin-depleted extracts (Cort D), or cortactin-depleted extracts supplemented with recombinant wild-type cortactin protein (Cort D + Cort), respectively. The mixtures were incubated at 37°C for 20 minutes. The cell pellets were treated with MesNa followed by quenching with iodoacetic acid and further analyzed for the presence of the remaining Biotin-SS-Tfn in the lysates, which represents MesNa resistance and transferrin internalized into CCVs. Data shown are the mean±s.d. of three experiments.
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