Raft containing fractions were tracked by the enrichment of the cholesterol bind ing protein, caveolin 1, and the dendritic lipid raft mar ker, flotillin 1. RT PCR and real time quantitative PCR analysis example The primer sets used in RT PCR for MIP 2��, GLT 1, GLAST, GFAP, and B actin were designed with Oligo soft ware. Total RNA was isolated from astrocytes using Trizol reagent. A total of 20 ug Inhibitors,Modulators,Libraries of RNA was reverse transcribed by using 200 U per ul of Moloney murine leukemia virus and 2 ug of random hex amer primers. Obtained templates were amplified in a final volume of 50 ul. Cyc ling conditions comprised an initial denaturation of 3 minutes at 94 C followed by 30 cycles of amplification and final elongation step at 72 C for 10 minutes in the presence of 20 Inhibitors,Modulators,Libraries pmol of primers.

Reac tion products were separated and visualized with eth idium bromide on a 1. 5% agarose gel. Real time PCR was performed in the Applied Biosystems 7500 Real Time PCR System software using SYBR GREEN PCR Master Mix. PCR was performed under the following conditions, initial denaturation at Inhibitors,Modulators,Libraries 95 C for 15 minutes and 37 cycles of 95 C for 30 seconds, 60 C for 30 sec onds, and 72 C for 20 seconds. The generation of specific PCR products was confirmed by melting curve analysis. Each reaction was run in triplicate. The ex pression of GLT 1 was normalized against B actin by the comparative threshold cycle method using the following formula, fold difference in expression Western blotting Whole cells were homogenized in radioimmunoprecipi tation assay buffer, 1% NP 40, 0.

Inhibitors,Modulators,Libraries 5% sodium deoxycholate and pro tein concentrations of the samples determined by the bicinchoninic acid method using BSA as a standard. Equal amounts of total protein were adjusted to similar volumes with loading buffer, denatured by heating at 95 C for 5 minutes, subjected to 10% SDS PAGE, and then electro blotted onto a nitrocellulose membrane using a minigel and mini transblot apparatus. The membranes were blocked with 5% nonfat dry milk in TBST buffer for 1 hour at room temperature. The blots were then incubated with either anti MIP 2��, anti GLT 1, anti GLAST, anti GFAP, anti flotilin 1, anti caveolin 1, or anti B actin antibodies diluted in TBS Tween overnight at 4 C. The blots were incubated with the appropriate horseradish peroxidase conjugated secondary antibodies for 1. 5 hours at room temperature.

Membrane bound Inhibitors,Modulators,Libraries second ary antibodies were detected using the chemiluminescence Super Signal procedure according to the manufac kinase inhibitor MEK162 turers instructions. Flow cytometric analysis of GLAST and GLT 1 Cells were washed twice with PBS, preincubated in PBS 1% BSA for 1 hour at 4 C, and then incubated with unconjugated rabbit polyclonal anti GLAST and anti GLT 1 antibodies for 1 hour at 4 C. Subsequently, cells were washed once with PBS 0. 1%BSA and stained with fluorescein isothiocyanate conjugated goat anti rabbit immuno globulin G for 30 minutes.