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Fig. 2. BPGAP1 interacts with EEN via its PRR. (A) BPGAP1 constructs used for identifying functional interactive domain for EEN. The NNP domain (aa 1-166), NP domain (aa 1-206), PC domain (aa 167-433) and full-length BPGAP1 were expressed as GST-recombinants in E. coli and affinity-purified with glutathione-sepharose beads. NNP, N-terminus with BNIP-2 and Cdc42GAP homology domain (BCH); NP, N-terminus with BCH domain and PRR; PC, C-terminus with GTPase-activating protein domain (GAP) and PRR. (B) BPGAP1 GST-recombinants were prepared and used for pull-down assays with 293T cell lysates as described in Materials and Methods. Blots were stripped and stained with amido black to reveal loading of GST-recombinants. Bound endogenous proteins were detected by EEN antibody. Lysates expressing Flag-EEN are used as positive controls and reference for the specificity of the EEN antibody used in detecting the endogenous EEN.





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