Neural Plasticity / 2011 / Article / Fig 6

Research Article

Diacylglycerol Signaling Underlies Astrocytic ATP Release

Figure 6

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 𝑛 = 4 , Munc13-1 siRNA = 4 , 𝑃 = 0 . 0 5 ). (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 𝑛 = 6 , Munc13-1 siRNA = 9 ; 𝑃 = 0 . 0 1 7 ).

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