Clinical, genetic and functional perspectives on ATP-binding cassette subfamily B member 4 variants in five cholestasis adults

Figure 5 Expression analysis and determination of transportation capability. A: MRNA expression analysis of ATP-binding cassette subfamily B member 4 (ABCB4)-wild-type (WT) and variants by qPCR. Using GAPDH as a reference for relative quantity analysis, Values were analyzed by GraphPad Prism 8.0. In addition, the results are the mean ± SD of four independent experiments. ^aP < 0.01, ^dP < 0.0001; B: Immunbloting of ABCB4 and variants. Representative western blot of the expression levels of the mutants with respect to ABCB4-WT. The expression and the processing of ABCB4-WT and the mutants was examined by western blot analysis of whole-cell lysates from transfected HEK293 cells. ABCB4 expression was detected following SDS-PAGE and immunoblotting with the anti- multidrug resistance protein 3 (MDR3) antibody. GAPDH was used as a loading control. Molecular masses are indicated on the left (in kDa). Mature MDR3 was show in black while immature MDR3 show in white; C: Western blot analysis of ABCB4-WT and variants. The values have been normalized to ABCB4-WT. And they were measured by ImageJ and analyzed by GraphPad Prism 8.0. The dates show the means ± SD of three separate experiments. ^bP < 0.01, ^cP < 0.001; D: Determination of transportation capability of MDR3 and its variants. The phosphatidylcholine levels in the supernatant of HEK293 cells were measured, which were respectively transfected with ABCB4-WT gene and mutant alleles. The secretion of phosphatidylcholine was standardized to wild type group. The data were analyzed by GraphPad Prism 8.0. And they represent the means ± SD of four independent experiments. ^bP < 0.01, ^dP < 0.0001. MDR3: Multidrug resistance protein 3; WT: ABCB4 wild type.