described above except without taxol. Velocity and Equilibrium Centrifugation Large membranes were removed from the mechanically permeabilized cell suspension by centrifugation at 1000 6 g. Gradients used iodixanol mixed with buffer B plus glutathione as media; continuous gradients were prepared using a two-chamber mixer. The supernatant of the 1000 6 g centrifugation was layered over 030% velocity gradients. For two dimensional separations, five velocity gradient fractions were collected, mixed with 60% iodixanol to a concentration of 32.5% or greater, and overlaid with a continuous 030% iodixanol/BB+G gradient and centrifuged to equilibrium. Refractive indices were measured using an Abbe refractometer and converted to density using the formula r = R.I.6 3.43193.5851, which was determined empirically by weighing known concentrations of iodixanol in buffer B. DRM Preparations Cracked cell suspensions were centrifuged at 1,000 6 g for 10 minutes. The P1 pellet was resuspended in buffer B containing protease inhibitors. Triton X100 or IGEPAL was added to a final concentration of 1% and the sample was vortexed and left on ice for at least 1 hour. The sample was centrifuged at 10,000xg for 10 MedChemExpress WP-1130 minutes to separate the detergent-soluble membranes from the detergent-insoluble pellet. This pellet was then resuspended in 180 ml BB with protease inhibitors and Triton X-100 was again added to a 1% concentration. Iodixanol was added to a 40% concentration and the samples sonicated for 2 6 5-second pulses. 
This step was necessary as in preliminary experiments without sonication, DNA and cytoskeletal elements often clogged the needle of the gradient fraction collector. Alternatively, instead of sonication, 2 ml Benzonase was added to the resuspended detergent resistant pellet and the sample incubated on ice for 1 hour to break up DNA prior to adding iodixanol. The method used for each set of experiments presented is identified in the figure legends. Samples were placed into 5 ml ultracentrifuge tubes and 10 40% or 1548% continuous iodixanol gradients in BB were poured over the top of the samples at 4uC. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189787 Gradients were centrifuged to equilibrium at 100,000xg for 1618 hours. Approximately 200 ml SDS-PAGE and Western Blotting SDS-PAGE gels were run and western blotted to nylonreinforced nitrocellulose as described. Where different treatment conditions are compared in one experiment, all blots were incubated in the TrkA in Microtubule-Rafts same antibody solution on the same day and exposed for the same amount of time. Blot incubations were performed in 5% nonfat dry milk, 150 mM NaCl, 50 mM Tris pH 7.7, 0.05% Tween 20, or conditions specified by the antibodies’ manufacturer. Secondary anti-mouse or rabbit antibodies coupled to HRP were used and chemiluminescent signals generated by Amersham ECLTM or Super Signal West Pico. The blot was either exposed to X-ray film or exposed directly in a Fujifilm Intelligent Dark Box II with a cooled CCD camera. Blots were stripped of antibodies for re-probing with Restore, TBS pH 2.0, or in 0.5 M NaCl, 0.2 M glycine, pH 2.8. the S1 and P1M samples were calculated by taking into account the volume that was loaded onto the gel compared with the original S1 and P1M sample size. Amounts of 125I-NGF in each fraction were determined using a gamma counter. For each protein, the amount in the floating DRM peak was calculated and expressed as a percentage of that particular protein in all cell fractions. P values were calculate
