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. 2021 Aug 9;6(15):e141395. doi: 10.1172/jci.insight.141395

Figure 8. TLR7 signaling is required for IRF5 phosphorylation, and IRF5 nuclear translocation is reduced in B cells from FcγRIIB−/−Yaa IRF5+/– mice.

Figure 8

(A and B) B cells were isolated from the spleens of FcγRIIB−/−Yaa mice at 8–10 weeks of age. (A) B cells were stimulated with anti-IgM, anti-CD40, and R848 alone or in combination for 2 hours and the protein lysate analyzed using phospho-Tag gel (upper panel) or standard gel (lower panels). B cells isolated from an IRF5-deficient (IRF5–/–) mouse are shown in the first lane. p-IRF5 denotes phosphorylated IRF5. A representative example of 7 individual experiments is shown. (B) Ratio of p-IRF5 to unphosphorylated IRF5 (u-IRF5). Intensity of p-IRF5 was normalized to the intensity of unphosphorylated IRF5 (lowest band of IRF5 on p-Tag gel as shown in A) (n = 7). (C and D) B cells from FcγRIIB−/−Yaa (WT) and FcγRIIB−/−Yaa IRF5+/– mice were stimulated for 2 hours with R848, or not stimulated (untreated), and IRF5 was probed in the nuclear and cytoplasmic fractions. (C) A representative experiment of 4 individual experiments is shown. (D) Ratio of IRF5 expression in nucleus relative to the WT after R848 stimulation; nuclear IRF5 intensity in each sample was first normalized to its own loading control (histone; n = 5). Data are shown as mean ± SEM and were analyzed using 1-way ANOVA with Tukey’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001. IRF5, IFN regulatory factor 5.