Figure 8. TrkA signaling endosomes associate with IC-2C dynein complexes in PC12 cells. (A) Colocalization of dynein and the TrkA growth factor receptor in PC12 cells. Neurite of a stable GFP–IC-2C PC12 cell treated with siRNA stained with dynein pan IC antibody, 74.1 (Dynein, green) and a pan Trk antibody (Pan Trk, red), and the overlay of the two colors. Arrowheads indicate some of the colocalizing puncta. (B) Colocalization of dynein and activated TrkA growth factor receptor in PC12 cells. Neurite of stable GFP–IC-2C cell line treated with siRNA stained with dynein pan IC antibody, 74.1 (dynein, green), and an antibody that reacts with activated (phosphorylated) Trk (pTrk, red). Arrowheads indicate some of the colocalizing puncta. Bar, 10 μm. (C) GFP–IC-2C–containing dynein is associated with TrkA containing organelles in PC12 cells. The membrane fraction (P2) was prepared from NGF-stimulated PC12 cells as described for Fig. 2, and GFP–IC–containing vesicles were isolated by immunoaffinity purification on magnetic beads laded with antibodies to GFP. The anti-GFP (α-GFP) and control IgG beads were washed, and the bound proteins were analyzed by SDS-Page and Western blotting with pan Trk antibody and the pan IC antibody, 74.1. The presence of GFP–IC (GFP–IC) on the beads was a positive control.
Figure 8.

TrkA signaling endosomes associate with IC-2C dynein complexes in PC12 cells. (A) Colocalization of dynein and the TrkA growth factor receptor in PC12 cells. Neurite of a stable GFP–IC-2C PC12 cell treated with siRNA stained with dynein pan IC antibody, 74.1 (Dynein, green) and a pan Trk antibody (Pan Trk, red), and the overlay of the two colors. Arrowheads indicate some of the colocalizing puncta. (B) Colocalization of dynein and activated TrkA growth factor receptor in PC12 cells. Neurite of stable GFP–IC-2C cell line treated with siRNA stained with dynein pan IC antibody, 74.1 (dynein, green), and an antibody that reacts with activated (phosphorylated) Trk (pTrk, red). Arrowheads indicate some of the colocalizing puncta. Bar, 10 μm. (C) GFP–IC-2C–containing dynein is associated with TrkA containing organelles in PC12 cells. The membrane fraction (P2) was prepared from NGF-stimulated PC12 cells as described for Fig. 2, and GFP–IC–containing vesicles were isolated by immunoaffinity purification on magnetic beads laded with antibodies to GFP. The anti-GFP (α-GFP) and control IgG beads were washed, and the bound proteins were analyzed by SDS-Page and Western blotting with pan Trk antibody and the pan IC antibody, 74.1. The presence of GFP–IC (GFP–IC) on the beads was a positive control.

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