zed to be due to increased TrkA association within lipid rafts,. Overexpression of the enzyme that produces GM1, however, has also been shown to GW788388 supplier decrease amounts of specific proteins associated with rafts and suppress TrkA dimerization, which is required for signaling activity. These data suggest that TrkA signal transduction causes its recruitment to lipid rafts. One possibility is that GM1 at very high levels may also dilute rafts or change the properties of the membrane such that signaling is impeded. To determine whether changes in lipid rafts affected the recruitment of TrkA Cell Fraction I-Ligand NGF Transferrin % total 1000 6 g pellet NP40 soluble NP40 insoluble 1000 6 g supernatant 67.2 4.3 30.4 SEM % total SEM 3.2 2.0 1.8 56.6 30.1 14.6 4.0 4.3 0.5 Cells were bound to radiolabelled ligand, washed, and subjected to internalization 10 min at 37uC. Mechanical permeabilization, fractionation, and detergent extraction was performed exactly as described. doi:10.1371/journal.pone.0035163.t001 TrkA in Microtubule-Rafts and p75NTR, we measured the effect of adding GM1 on the amount of NGF, its receptors, and microtubules in DRMs. GM1 increased NGF and Trk in DRMs more than 2-fold. In contrast, p75NTR and flotillin were affected by GM1 in the opposite way. GM1-treated cells had less than half the amounts of p75NTR and flotillin in floating DRMs compared to those of control. It is important to note that the fraction of p75NTR and flotillin in DRMs is constitutively high, about 20% without GM1 treatment, compared to TrkA. p75NTR and flotillin are known to preferentially associate with lipid rafts in many different cell types, and this property may be related to their 
similar decrease in DRMs in GM1-treated cells. The data are consistent with high levels of GM1 diluting rafts, which affects proteins that preferentially or constitutively associate with rafts differently than proteins that transiently associate with rafts in response to stimulation. We also found that the microtubules that associated with floating DRMs increased more than 3-fold after GM1 treatment. Thus, GM1’s effects, as with PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189787 in vitro reactions that cause microtubules to polymerize, were to increase microtubules in DRMs, which correlated with increases in NGF and TrkA. In both cases p75NTR behaved in the opposite manner. The data suggest that NGF is mostly bound to TrkA, not p75NTR, TrkA in Microtubule-Rafts in floating DRMs, because the changes in the distribution of NGF paralleled that of TrkA. We used two different methods to break up the insoluble material in the detergent-resistant pellet: sonication and nuclease treatment. When sonication was used, the density of the floating peak was approximately 1.16 g/ml. Trk was associated with two peaks on these gradients, one of which coincided with the floating NGF peak. The other peak was of higher density and did not coincide with NGF. p75NTR was also found in the 1.16 g/ml floating peak with NGF and Trk, and little was present in other regions of the gradient. A fraction of tubulin also remained in the non-floating bottom of the gradient under these conditions. These data indicate that a fraction of the microtubules in DRMs were reproducibly resistant to sonication. To further investigate this possibility we determined whether results could be obtained without sonication. We found that Benzonase treatment facilitated handling of the DRM fraction without sonication. Quantitative distribution of receptors into floating DRMs w
