Fig 2: Mechanism of regulated binding of an NLS in GFP–capicúa to KPNA3(A) HEK-293 cells were transfected to express GFP–capicúa (GFP–CIC) and GFP–capicúa with a double R1412A and K1413A substitution in the putative NLS [GFP–CIC (NLS*)], and stimulated or not with EGF±PD184352. Lysates were subjected to GFP–Trap® immunoprecipitation, and immunoprecipitates and whole-cell lysates were immunoblotted with the antibodies indicated. (B) HEK-293 cells were transfected to express the indicated GFP–capicúa proteins and stimulated or not with EGF. Cell lysates and GFP–Trap® immunoprecipitates were analysed with the antibodies indicated. (C) HEK-293 cells were transfected to express GFP–capicúa and cell lysates were incubated with the indicated concentrations of synthetic wild-type (CASPKKKRKV-OH) or mutant (CASPKTKRKV-OH) simian-virus-40 large T-antigen NLS peptides for 30 min at 4 °C followed by GFP–Trap® immunoprecipitation and analysis by immunoblotting. (D) GFP–capicúa and HA–KPNA3 were expressed separately in HEK-293 cells that were stimulated or not with EGF±PD184352. Different combinations of the lysates were mixed as indicated, and GFP–capicúa was immunoprecipitated from the mixtures and immunoblotted with the antibodies indicated. (E) HEK-293 cells were transfected to express wild-type and mutated forms of GFP–capicúa as indicated and stimulated or not with EGF±PD184352 followed by GFP–Trap® immunoprecipitation. Immunoprecitated proteins were analysed by Western blotting using anti-GFP and anti-KPNA3 antibodies. (F) DAPI staining and GFP immunofluorescence of HEK-293 cells transfected with the indicated GFP–capicúa constructs. Scale bar=10 μm. IB, immunoblot.

Fig 3: Identification of regulated phosphorylated sites on GFP–capicúa and its regulated interactions with KPNA3 and 14-3-3(A) GFP–capicúa (CIC) and GFP (control) proteins were isolated from lysates of transfected cells using GFP–Trap® (an immobilized single-chain antibody) coupled to agarose. Cells were stimulated with EGF (100 ng/ml for 30 min) and PMA (100 ng/ml for 30 min) with or without pre-incubation with the p90RSK inhibitor BI-D1870 (10 μM for 30 min). Captured proteins were subjected to SDS/PAGE, gels were stained with colloidal Coomassie Blue, and proteins and phosphorylated sites were identified by MS as described in the Materials and methods section and Supplementary Figure S3 (at http://www.BiochemJ.org/bj/433/bj4330515add.htm). Arrows point to the proteins identified as GFP–capicúa (GFP–CIC), 14-3-3s and KPNA3. The molecular mass in kDa is indicated on the left-hand side. (B) Effects of inhibitors of the ERK pathway and p90RSK on the EGF-regulated binding of KPNA3 and 14-3-3 to GFP–capicúa. HEK-293 cells were transfected to express GFP–CIC (full-length, WT) and GFP–CIC-(1–450), and stimulated or not with EGF±PD184352 (2 μM inhibitor for 60 min) and±BI-D1870 (10 μM inhibitor for 30 min). EGF was used at 100 ng/ml for 30 min. Lysates (2.5 mg) were subject to GFP–Trap® immunoprecipitation, followed by immunoblotting with the antibodies indicated. In addition, the ability of the GFP–CIC-(1–450) to bind directly to 14-3-3 was tested with a 14-3-3 Far-Western assay. IB, immunoblot.

Fig 4: Capsid interacts with other importin α family members. Following infection with VEEV V5-C (MOI 1), HMC3 cells were collected, protein lysates were quantified, and subjected to co-immunoprecipitation assays. A hemagglutinin (HA) antibody pulldown was included as a co-immunoprecipitation control. (A) Immunoprecipitation of V5-tagged capsid was done using a V5 tag antibody and the blot was probed with either a KPNA3 (importin α4) antibody or V5 antibody. (B) Immunoprecipitation of V5-tagged capsid was done using a V5 tag antibody and the blot was probed with either a KPNA4 (importin α3) or V5 antibody.