Figure 3: IRF5 expression is directly regulated by IRF8.
From: Transcription factor IRF5 drives P2X4R+-reactive microglia gating neuropathic pain
Real-time PCR analysis of Irf5 mRNA in BV2 cells transduced with (a) IRF8-GFP (high or low dose of IRF8) or GFP alone (control) (n=8; ***P<0.001), or (b) IRF8-GFP (IRF8), IRF8(K79E)-GFP (IRF8(K79E)) or GFP alone (control) (n=8; ***P<0.001). Representative western immunoblot of IRF5 (of three independent experiments) in BV2 cells transduced with (c) IRF8-GFP (high or low dose of IRF8) or GFP alone (control), or (d) IRF8-GFP (IRF8), IRF8(K79E)-GFP (IRF8(K79E)) or GFP alone (control). (e) Real-time PCR analysis of Irf5 mRNA in primary cultured microglia transduced with IRF8-GFP (IRF8), IRF8(K79E)-GFP (IRF8(K79E)) or GFP alone (control) (n=6; ***P<0.001). (f) Schematic of the predicted two binding sites of IRF8 on the promoter region of IRF5. (g) ChIP-qPCR assay of Irf5 promoter fragments immunoprecipitated by antibodies for IRF1, IRF3, IRF7, IRF8 or IRF9 in BV2 cells transduced with IRF8-GFP or normal BV2 cells. Values represent the relative ratio of the values of BV2 cells transduced with IRF8-GFP (normalized to the value for normal IgG) to that of normal BV2 cells. Data are representative of three experiments. (h) Luciferase activity of BV2 cells transduced with Irf5 reporter gene, plus IRF8-GFP (IRF8) or GFP alone (control) (n=8). (i) Real-time PCR analysis of Irf5 mRNA in the spinal cords of WT and Irf8−/− mice 7 days after PNI. Values represent the relative ratio of Irf5 mRNA (normalized to the value for 18s mRNA) to the contralateral side of WT mice (n=7; ***P<0.001). (j) Upper: representative western immunoblots of IRF5 in the spinal cords of WT and Irf8−/− mice 7 days after PNI. Lower: relative band density ratios of IRF5 (normalized to β-actin) to the contralateral side of WT mice (n=6; ***P<0.001). (k) Real-time PCR analysis of Irf8 mRNA in total RNA extracted from the spinal cords of WT and Irf5−/− mice 7 days after PNI. Values represent the relative ratio of Irf8 mRNA (normalized to the value for 18s mRNA) to the contralateral side of WT mice (n=7; ***P<0.001). Values are the mean±s.e.m. Full-size blots are shown in Supplementary Fig. 10.
