Fig. 6

Assay of the efflux capacity of 8-[Fluo]-cAMP in HEK293T cells expressing hABCC4. To investigate the binding flexibility and tolerance of hABCC4 toward cAMP, as well as the potential of 8-[Fluo]-cAMP as a detection tool in transport assays, we designed cellular transport experiments to examine the impact of key residue mutations involved in hABCC4 recognition and binding of cAMP on the transport of 8-[Fluo]-cAMP. a The molecular structures of cAMP, 8-[Fluo]-cAMP, and FTSC are presented. The fluorescent tag regions, which may influence hABCC4 recognition, are highlighted by red dashed lines. b After incubation with 8-[Fluo]-cAMP and FTSC, cell lysate supernatants from cells expressing hABCC4-WT or the empty vector pCDNA3.1 are shown. Each sample shown in the figure was randomly selected from four parallel samples in the corresponding experimental group. The upper panel shows the original images, whereas the lower panel shows images with adjusted brightness to enhance the visibility of green fluorescence. c Fluorescence intensity of the cell lysates from (b) was measured via a fluorescence microplate reader (n = 4; data are presented as the means ± SEMs). The results demonstrated that cells transfected with hABCC4-WT exhibited significant 8-[Fluo]-cAMP efflux activity, whereas no significant transport activity was observed for FTSCs. d Western blot analysis (WB) of hABCC4 protein expression in cell lysates from the 8-[Fluo]-cAMP and FTSC transport experiments. e Cell lysates from HEK293T cells expressing various hABCC4 residue mutants after incubation with 8-[Fluo]-cAMP. f Fluorescence intensity of the cell lysates from (e) was measured via a fluorescence microplate reader (n = 4; data are presented as the means ± SEMs). g Western blot analysis (WB) of hABCC4 protein expression in cell lysates from cells transfected with various hABCC4 residue mutants