Diacylglycerol Signaling Underlies Astrocytic ATP Release
The Munc13-1 protein is expressed in cultured astrocytes and reduction of Munc13-1 expression reduces astrocytic ATP release. (a) Immunocytochemistry for Munc13-1 in cultured astrocytes demonstrates expression of Munc13-1 in these cells: (green: Munc13-1, blue: DAPI). (b) Western blotting for Munc13-1 demonstrates a band at the expected size (~200 kD). Gel lanes were loaded with 60 μg of cultured mouse astrocyte protein or 15 μg of protein extracted from cultured mouse cortical neurons. (c) RT-PCR with primers designed against Munc13-1 of whole mouse brain mRNA and mRNA extracted from cultured astrocytes demonstrated a band for Munc13-1 at the expected size. Sequencing of these bands confirmed the presence of Munc13-1 in cultured astrocytes. (d) siRNAs designed against mouse Munc13-1 reduce the expression of Munc13-1 in transfected mouse astrocytes as measured by Western blot. Optical density of the Munc13-1 signal was standardized against the housekeeping protein GAPDH (scrambled siRNA , Munc13-1 siRNA ). (e) The response of cultured astrocytes to OAG during one imaging session was measured for cultured astrocytes transfected with either scrambled control siRNAs or siRNAs directed against the Munc13-1 mRNA. The percentage of cells responding to OAG was significantly reduced after transfection with Munc13-1 siRNA (scrambled siRNA , Munc13-1 siRNA ).
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