Figure 1.

Astrocyte-specific expression of dnEGFR blocked injury-induced ErbB activation in reactive astrocytes. (a) Representative images of TRE-YFP expression in astrocytes of Mlc1-tTA mice at 1-month old. AAV-TRE-YFP was stereotaxically injected into indicated brain regions. Fixed brains collected 1 day later were sectioned and immunostained for Acsbg1 or GFAP to label astrocytes in the cerebral cortex or hippocampal hilus, respectively. White arrows, double-positive cells. (b) Efficient inhibition on activity of each ErbB receptor by dominant-negative mutant dnEGFR. Complementary DNA sequence of dnEGFR was amplified from genomic DNA of TRE-dnEGFR mice by PCR and cloned into pcDNA3.1-His/myc vector. pcDNA3.1-dnEGFR-myc was transfected into HEK293 cells by polyethylenimine (PEI) together with one of ErbB1–4 plasmids or an empty vector. Cells were lysed 24 h later and processed into WB with indicated antibodies. Any ErbB receptor when overexpressed in HEK293 cells would autophosphorylate itself independent of ligand stimulation. Shown are representative WB results, demonstrating the inhibition of dnEGFR on phosphorylation of each ErbB receptor. (c) Quantitative analyses of experiments in (b). ***P<0.001; **P<0.01; n=3 for each ErbB receptor, paired t-test. (d) Schematic illustration of the Tet-off system in Mlc1-tTA;TRE-dnEGFR (Mlc1-dnEGFR) mice. (e, f) Active ErbB3 (pErbB3), but not total ErbB3 levels, was suppressed in the reactive astrocytes of Mlc1-dnEGFR cortex. Cortical slices from indicated mice 3 days post injury were immunostained with antibodies against pErbB3 (e) or total ErbB3 (f) together with GFAP. White arrows, double positive cells. Arrowheads, cells positive for GFAP alone.