Figure 3. IRTKS associates with PCBP2.

(a) IRTKS interacts with PCBP2 by yeast two-hybrid screening. Yeast strain AH109 was co-transfected with Gal4 DNA-binding domain (BD)-fused IRTKS and Gal4 activating domain (AD)-fused PCBP2. p53 and large T antigen were introduced as a positive control. (b) Recombinant GST-IRTKS and PCBP2 were subjected to GST pull-down assay. (c) WT BMDMS were infected with VSV (m.o.i.=5) for 2 h, followed by immunoprecipitation (IP) with anti-IRTKS antibody. (d) IRTKS^+/+ and IRTKS^−/− mice were intranasally inoculated with VSV (5 × 10⁵ p.f.u. for each mouse) for the indicated times. Peritoneal macrophages were collected and lysed for IP with anti-IRTKS antibody. (e) WT BMDMS were infected with HSV (m.o.i.=5) for 2 h, followed by IP with anti-IRTKS antibody. (f) Scheme for IRTKS truncations. (g) The indicated Flag-tagged IRTKS truncations were co-transfected with FL HA-tagged PCBP2 into MEF cells, followed by IP with anti-Flag antibody. (h) IRTKS^−/− BMDMs were rescued with FL- or Δ400–514-IRTKS, followed by infection with VSV-GFP (m.o.i.=5) for 24 h. Cells were counterstained with DAPI (left panel). GFP-positive cells were calculated (right panel). Scale bar, 200 μm. (i,j) The indicated Flag-tagged PCBP2 truncations (i) were co-transfected with FL HA-tagged IRTKS into MEF cells, followed by IP with anti-Flag antibody (j). (k) PCBP2-silenced BMDMs were rescued with FL- or Δ1–81-PCBP2, followed by infection with VSV-GFP (m.o.i.=5) for 24 h. Cells were counterstained with DAPI (left panel). GFP-positive cells were calculated (right panel). Scale bar, 200 μm. Data are shown as means±s.d. A two-tailed unpaired Student's t-test was used. **P<0.01; ***P<0.001. Data are representative of at least three independent experiments. m.o.i., multiplicity of infection; p.f.u., plaque-forming unit.