Using this model, we observed similar increases in dnEGFR transcripts in injured and intact Mlc1-dnEGFR cortices (Supplementary Figure S3c and d). To investigate whether ErbB receptor activity was altered in the reactive astrocytes of Mlc1-dnEGFR mice, injured cortical slices were costained with antibodies to GFAP and the active forms of ErbB1–4. Although other antibodies did not detect signals, immunoreactivity of phosphorylated ErbB3 (pErbB3) was clearly observed in the reactive astrocytes of control mice (Figure 1e). Notably, pErbB3 was hardly detected in the reactive astrocytes of Mlc1-dnEGFR mice, indicating inhibition of endogenous ErbB signaling by dnEGFR (Figure 1e). Note non-detection of pErbB3 was not due to loss of the ErbB3 protein because ErbB3 immunoreactivity was present in the reactive astrocytes of Mlc1-dnEGFR mice in a manner similar to that observed in controls (Figure 1f). The involvement of other ErbB receptors in reactive astrocytes could not be excluded, as their absence could have been due to the used assays being insensitive to small amounts of protein. In order to test this idea, we cultured primary astrocytes isolated from TRE-dnEGFR or Mlc1-dnEGFR mouse brains. All four ErbB receptors were detected in the primarily cultured astrocytes by Western blotting (WB) (Supplementary Figure S3e). We treated the primary astrocytes with recombinant human EGF (rhEGF), a ligand specifically binding to EGFR, and revealed activation of all four ErbB receptors in control astrocytes (Supplementary Figure S3e and f). Unsurprisingly, ErbB receptor activities induced by rhEGF were significantly reduced in primary Mlc1-dnEGFR astrocytes (Supplementary Figure S3f). Treatment with recombinant human NRG1, another ligand that specifically binds to ErbB3 or ErbB4, induced ErbB4 activation in primary control astrocytes. Remarkably, it was significantly reduced in primary Mlc1-dnEGFR astrocytes (Supplementary Figure S3e and f). These results consolidated the inhibitory effects of dnEGFR on ligand-induced ErbB receptor activation. It was noticeable that basic activities of EGFR, ErbB2 and ErbB3 were suppressed in primary Mlc1-dnEGFR astrocytes (Supplementary Figure S3f), suggesting an inhibition on efficiency of endogenous ErbB ligands. Therefore, dnEGFR targeting of ErbB receptor kinase activity helped us circumvent the limited knowledge of ErbB receptor composition in vivo and focus on characterizing their function in reactive astrocytes.