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. 2014 May 13;5:3771. doi: 10.1038/ncomms4771

Figure 2. IRF5 is required for fibronectin-mediated upregulation of P2X4R in microglia.

Figure 2

(a) Representative western immunoblot of IRF5 in whole-cell lysate, and in nuclear or cytosolic components of control or IRF5 shRNA-transduced BV2 cells treated with fibronectin for 4 h. (b) Relative band density ratios of IRF5 (normalized to β-actin, lamin B or α-tublin) from control or IRF5 shRNA-transduced BV2 cells treated with fibronectin (n=4; *P<0.05, ***P<0.001). (c) Real-time PCR analysis of P2rx4 mRNA in control or Irf5 shRNA-transduced BV2 cells 6 h after fibronectin treatment. Values represent the relative ratio of P2rx4 mRNA (normalized to the value for 18s mRNA) to control shRNA-transduced cells (n=6; *P<0.05, **P<0.01, ***P<0.001). (d) Representative western immunoblot of P2X4R in control or IRF5 shRNA-transduced BV2 cells 6 h after fibronectin treatment. (e) Relative band density ratios of P2X4R (normalized to β-actin) to control shRNA-transduced cells (n=6; ***P<0.001). (f) Schematic of the four interferon-stimulated response element (ISRE) sites on the promoter region of P2X4R. (g) Chromatin immunoprecipitation (ChIP)-qPCR assay of P2rx4 promoter fragments immunoprecipitated by antibodies for IRF1, IRF3, IRF5, IRF7 or IRF9 in BV2 cells with or without fibronectin, respectively. Values represent the relative ratio of the values of BV2 cells with fibronectin (normalised to the value for normal IgG) to that of cells without fibronectin. Data are representative of three experiments. Values are the mean±s.e.m. Full-size blots are shown in Supplementary Fig. 9.