Figure 2

Localization of ABCG2 in human skin by western blot, RT-PCR assay and flow cytometry analysis. (A) Western blot analysis of ABCG2 expression in human skin. Epidermis and positive control (A549 cell line) expressed ABCG2, dermis tissue did not; a lower level of ABCG2 staining was detected in whole unfractionated skin. (B) RT-PCR analysis of ABCG2 mRNA expression in sorted keratinocytes. Higher expression of ABCG2 mRNA (379 base pairs (bp)) was shown in ABCG2-positive keratinocytes sorted by fluorescence-activated cell sorting, while lower expression of ABCG2 mRNA was shown in ABCG2-negative keratinoctyes. A 100 bp DNA ladder is shown in the first left lane. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 498 bp) was used as an internal control. (C) Fresh isolated epidermal keratinocytes were incubated with anti-ABCG2 antibody (clone 5D3), and Alexafluor-488 conjugated secondary antibody was used. ABCG2-positive cells (green line, region M1) accounted for approximately 2.7%; isotype control, blue line; blank control, red line. (D) Primary cultured epidermal keratinocytes were harvested and incubated with anti-ABCG2 antibody; approximately 2.8% of cells showed ABCG2-positive staining (green line, region M1). The flow analyses were repeated five times, and the results were averaged.