as similar after sonication or Benzonase treatment, but the receptors floated into a peak of slightly higher density and there was less non-floating material in benzonase-treated samples. Actin filaments were also present in floating DRMs under these conditions, but there was no change in their association with DRMs after in vitro reactions. Importantly, comparable increases in NGF and microtubules in floating DRMs after in vitro reactions were observed. TrkA was reproducibly dephosphorylated in floating DRMs. Under conditions where phospho-TrkA was detected in the detergent-sensitive fraction, and in endosomes, TrkA but not pTrkA was present in floating DRMs. The presence of the tyrosine phosphatase, SHP-1 in floating DRMs suggests a mechanism by which TrkA is selectively dephosphorylated in this fraction. The similar increases in NGF, TrkA 
and microtubules in DRMs in response to GM1 and in vitro reactions suggest that TrkA may bind to microtubules in this fraction. Indeed, TrkA was coprecipitated when microtubules were stabilized with taxol and immunoprecipitated from floating DRMs. If biotinylated tubulin was added during the last 5 min of in vitro reactions, it was incorporated into floating DRMs, suggesting that newly polymerized microtubules were associated with this fraction. Streptavidin MedChemExpress SR 2516 agarose beads recovered TrkA from this fraction and biotinylated tubulin was pulled down by TrkA immunoprecipitation. p75NTR was not reproducibly detected in microtubule immunoprecipitations in these experiments. These data suggest that microtubule polymerization and attachment to DRMs recruits TrkA. Under these conditions, biotinylated tubulin accumulated in discrete foci at the plasma membrane of permeabilized cells 4 TrkA in Microtubule-Rafts along with microtubules, but not p75NTR or flotillin, indicate that sorting specificity is reconstituted. NGF Affected the Amount of its Receptors in Rafts We compared the effects of NGF on TrkA and p75NTR in floating DRMs. Without in vitro reactions, NGF caused a 1.5- to 2fold increase of both TrkA and p75NTR in the floating peak. In contrast, after in vitro reactions, NGF caused TrkA to increase, and p75NTR to decrease in the floating DRMs. It has been noted previously that NGF signaling enhances tubulin PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22187495 polymerization. These data, together with that showing that microtubules assemble and associate with floating DRMs during in vitro reactions, suggest that NGF signaling may enhance microtubule association with DRMs during these reactions. Indeed, NGF significantly increased amounts of microtubules in floating DRMs, which correlated with increased TrkA in this fraction. In contrast, p75NTR was significantly reduced in floating DRMs after NGF treatment under these conditions, and flotillin was unchanged by NGF. These results indicate that NGF differently affected localization of the two co-receptors in floating DRMs under conditions where microtubules assemble and associate with membranes. TrkA in Detergent Resistant Endosomal Fractions One possible outcome of sorting in rafts could be for conveying receptors into different endosomes. We asked whether TrkA could be detected in lipid rafts associated with microtubules in endosomes. We examined endosomes using organelle fractionation methods described previously. Organelles that emerged from mechanically permeabilized cells were subjected to velocity sedimentation followed by floatation equilibrium centrifugation on iodixanol gradients. Endosomes containin
