Figure 1: PNI induces the expression of IRF5 in spinal microglia.

(a) In situ hybridization analysis of Irf5 mRNA in the spinal dorsal horn of mice 7 days after PNI. Black arrowheads indicate Irf5 mRNA signals. Images are representative of three experiments. Ipsi, ipsilateral; Contra, contralateral. (b) Representative images (of three experiments) showing that Irf5 mRNA signals colocalize with the immunoreactivity of Iba1 (black arrowheads), but not with that of GFAP (red arrowheads). ISH, in situ hybridization; IHC, immunohistochemistry. (c) Real-time PCR analysis of Irf5 mRNA in total RNA extracted from spinal cord ipsilateral and contralateral to PNI before (naive) and after PNI. Values represent the relative ratio of Irf5 mRNA (normalized to the value for 18s mRNA) to the contralateral side of naive mice (n=6; ***P<0.001). (d) Representative western immunoblot of IRF5 protein in homogenates from the ipsilateral and contralateral spinal cords of wild-type (WT) mice before (naive) and after PNI. (e) Relative band density ratios of IRF5 (normalized to β-actin) to the contralateral side of naive mice at each time point (n=6; **P<0.01, ***P<0.001). Values are means±s.e.m. Full-size blots are shown in Supplementary Fig. 8. Scale bar, 200 μm (a, upper), 30 μm (a, lower), 30 μm (b).