RT PCR analysis revealed that buffer definitely treated astrocytes expressed messages for the housekeeping gene, B actin, and traces of MIP 2��, whereas LPS and TNF treat ment increased MIP 2�� expression. Inhibitors,Modulators,Libraries Immunoblot analysis also showed that LPS and TNF increased MIP 2�� protein expression in astrocytes. Specific knockdown MIP 2�� expression in cultured astrocytes by siRNA We first examined the efficiency of astrocyte transduc tion using a pAAV MIP 2�� hrGFP vector, and at least 80% produced robust green fluorescence within two to three days that did not diminish. We then used a siRNA to block MIP 2�� expression. Astrocytes were cultured on 6 well plates and transfected with either MIP 2�� siRNAs or the pBS U6 con vector together with pAAV MIP 2�� hrGFP 24 hours after plating.
At 48 hours post transfection, cell extracts were assayed by immunoblotting for MIP 2�� protein. Transfection with siRNAs directed at specific MIP 2�� mRNA sequences suppressed MIP 2�� protein, es pecially MIP 2�� siRNA 1, whereas transfection with the pBS U6 con did not. In addition, transfection with MIP 2�� siRNAs did not dramatically change levels of GFAP, a Inhibitors,Modulators,Libraries marker for astrocyte activation, suggesting that astrocyte function was not disrupted. MIP 2�� decreases astrocyte expression of GLT 1 Treatment of astrocytes with dBcAMP increases both GLT 1 and GLAST expression. Our astrocyte cul tures are routinely cultured with 250 uM dBcAMP, so GLAST and GLT 1 are both expressed. Transfection with pAAV MIP 2�� hrGFP reduced GLT 1 expression but not GLAST, and this decrease could be partly reversed by MIP 2�� siRNA 1 .
Flourescence activated Inhibitors,Modulators,Libraries cell sorting analysis showed that transfection of pAAV MIP 2�� hrGFP decreased cell surface GLT 1 expression, which was again partly reversed by MIP 2�� siRNA 1, but did not affect GLAST expression. Western blots also showed that MIP 2�� overexpression decreased total cellular GLT 1 expression but not GLAST expression. The actin band provides an index of intracellular proteins present in each preparation, and the cytoskeletal protein, GFAP, confirms the cells are astrocytes. GLT 1 molecules are Inhibitors,Modulators,Libraries organized on lipid rafts in astro cytes, which may be necessary for efficient glutamate up take. To isolate these rafts, we performed sucrose gradient ultracentrifugation of detergent free cell extracts of MIP 2�� transfected cells in the presence or absence of MIP 2�� siRNA 1 plasmids.
Immunoblots of pooled fractions corresponding to the raft domains, as confirmed by the presence of the protein, flotillin 1, and Inhibitors,Modulators,Libraries detergent soluble material showed that both GLAST and GLT 1 were recovered in rafts. MIP 2�� overex pression selleck kinase inhibitor reduced levels of GLT 1 in the raft domains, but did not affect GLAST recruitment into raft domains. Interestingly, MIP 2�� overexpression in creased caveolin 1 levels but did not affect flotilin 1 levels, suggesting that the down regulation of GLT 1 was specific.
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