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. 2015 Sep 8;6:8132. doi: 10.1038/ncomms9132

Figure 6. IRTKS-mediated PCBP2 sumoylation causes its cytoplasmic translocation leading to MAVS degradation.

Figure 6

(a) IRTKS+/+ and IRTKS−/− BMDMs were incubated with VSV (m.o.i.=5) for 8 h with or without ginkgolic acid (GA) (20 μm), followed by immunostaining with the indicated antibodies. Percentages of cytoplasmic PCBP2 (cytoplasmic PCBP2/total PCBP2) were calculated (right panel). At least 200 cells were counted. Scale bar, 10 μm. (b) WT BMDMs were incubated with VSV (m.o.i.=5) for 8 h, followed by immunoprecipitation (IP) with anti-PCBP2 antibody using cytoplasmic or nuclear fractions. (c) WT BMDMs were incubated with VSV (m.o.i.=5) for 8 h with or without GA (20 μm), followed by cytoplasmic and nuclear separation. IP was performed using anti-PCBP2 antibody. Immunoprecipitates were detected with the indicated antibodies. (d) PCBP2+/+ and PCBP2−/− BMDMs rescued with WT or K37R-PCBP2 were incubated with VSV (m.o.i.=5) for 8 h, followed by immunostaining with the indicated antibodies. Percentages of cytoplasmic PCBP2 (cytoplasmic PCBP2/total PCBP2) were calculated (right panel). At least 200 cells were counted. Scale bar, 10 μm. (e) PCBP2+/+ and PCBP2−/− BMDMs rescued with the indicated PCBP2 plasmids were incubated with VSV (m.o.i.=5) for 8 h, followed by IP with anti-PCBP2. (f) PCBP2+/+ and PCBP2−/− MEFs were transfected with Flag-AIP4 and Myc-PCBP2 (K37R) for 24 h, followed by immunoblotting with the indicated antibodies. (g) IRTKS+/+ and IRTKS−/− MEFs were transfected with Flag-AIP4 for 24 h with or without 40 nM leptomycin B, followed by immunoblotting with the indicated antibodies. (h) shCtrl- or AIP4-silenced MEFs were transfected with Flag-IRTKS for 24 h, followed by immunoblotting with the indicated antibodies. Data are shown as means±s.d. Data are representative of at least three separate experiments. m.o.i., multiplicity of infection.