Fig. 5.

ADAP competes with STAT1 for importin α5 binding. A Importin α5 interacts with both ADAP and STAT1 in response to IFN-γ in RAW264.7 cells. RAW264.7 cells were stimulated with IFN-γ for the indicated time points, and the interaction between importin α5 and ADAP or phosphor-STAT1 was determined by immunoprecipitation with anti-importin α5, followed by immunoblotting with antibodies against ADAP and pY701-STAT. B The effects of ADAP deficiency on the interaction between STAT1 and importin α5. WT or ADAP-KO RAW264.7 cells were treated with or without IFN-γ as indicated. The interaction between STAT1 and importin α5 was determined by immunoprecipitation with anti-importin α5, followed by immunoblotting with pY701-STAT1 antibody. C Immunoprecipitation and immunoblot analysis of the ADAP/STAT1 interaction in control or importin α5 KO RAW264.7 cells with or without IFN-γ stimulation. D Competitive in vitro binding of ADAP and STAT1 to importin α5 by GST pulldown assay. HEK 293 T cells were transfected with equal amounts of FLAG-STAT1 constructs and increased amounts of HA-ADAP constructs. Cell lysates were incubated with equal amounts of purified GST-importin α5 (aa 400–538) protein, followed by pull-down with GST-tag purification resin. The precipitates were subjected to immunoblot analysis with anti-HA and anti-FLAG antibodies. E GFP-importin α5 was coexpressed with empty vector, HA-ADAP WT, or HA-ADAP truncation mutants in HEK 293 T cells. Cell lysates were subjected to immunoprecipitation using anti-GFP antibody, followed by blotting with anti-HA and anti-FLAG antibodies. F Surface plasmon resonance analysis of the purified importin α5 (aa 400–538) interactions with ADAP NLS1, NLS2 or STAT1 NLS peptide