Regulation of dynein by neurotrophins and Trks. (A) Recruitment of activated (phosphorylated) TrkA to dynein containing vesicles in NGF-treated PC12 cells. PC12 cells were harvested and then treated with NGF (+NGF) or buffer (−NGF). The postnuclear supernatant (S1) and the membrane fraction (P2) were prepared and analyzed by SDS-PAGE and Western blotting. The blot was first probed with an antibody specific for the phosphorylated (activated) form of Trk (pTrk). Next, the blot was stripped and probed with pan Trk antibody (panTrk). The vesicle fractions were immunoprecipitated (IP) with anti-pan IC antibody (74.1) or control IgG (IgG) on magnetic beads, and a Western blot of the proteins bound to the beads was probed with the antibody to activated Trk (pTrk). The blots were probed with a pan Trk antibody as a loading control. (B) Modulation of dynein by NGF. (left) Graphs of mean anterograde (blue) and retrograde (red) interval velocities for control (Control) and NGF-treated (+NGF) PC12 cells. The mean interval retrograde velocity for NGF-stimulated cells was 1.2 μm/s ± 0.05 standard error (n = 140), and for control cells 0.9 μm/s ± 0.03 standard error (n = 300). The difference in mean retrograde velocities was significant at P < 0.00017 (Student's t test). (right) Graph showing the percent of GFP–IC-2C dynein puncta moving in the anterograde (blue) and retrograde (red) directions in control (Control) and NGF-stimulated cells (+NGF). The addition of NGF resulted in a significant increase in the relative number of dynein puncta moving in the retrograde direction (P < 0.0002, χ² test).