Figure 3.

TrkA is transactivated on internal membranes. A, A receptor insertion assay was performed as described in Materials and Methods and is depicted in the schematic diagram. PC12 cells were treated with the biotinylation-blocking reagent S-NHS-acetate and then treated with either 10 nm CGS 21680 or 100 ng/ml NGF for 3 hr. Cell lysates were immunoprecipitated with avidin-agarose. Supernatants from the avidin-agarose immunoprecipitation that were depleted of biotinylated material were subsequently subjected to immunoprecipitation with pan-Trk antibodies. B, Immunoprecipitates were analyzed by blotting with phosphotyrosine antibodies, antibodies to phosphorylated Trk, or pan-Trk antibodies. Untreated cells that were biotinylated immediately after block (lane 1) or 3 hr after block (lane 2) were used as controls for basal levels.