Fig. 5
From: Structural basis of human ABCC4 recognition of cAMP and ligand recognition flexibility
Impact of hABCC4 Residue Mutations on MTX Transport. To confirm whether the poorly distributed density in the W995-R998 region of the binding pocket in the 9KRM structure corresponds to the MTX ligand, we designed transport assays to evaluate the impact of the relevant residues on MTX binding. a Activity of HEK293T cells transfected with ABCC4-WT, ABCC4-W995A, or pCDNA3.1-EGFP after exposure to different concentrations of MTX for 48 h (data are presented as the means ± SEMs). This result revealed that the optimal MTX concentration for residue mutation transport experiments is 100 nmol/L. b Western blot results showing hABCC4 protein expression in HEK293T cells transfected with ABCC4-WT, ABCC4-W995A, or pcDNA3.1-EGFP. c Activity of HEK293T cells transfected with hABCC4 mutant vectors in the presence of 100 nM MTX (data are presented as the means ± SEMs). d Western blotting results demonstrating protein expression in HEK293T cells transfected with mutant hABCC4 vectors