Null alleles of ABCG2 are responsible for the Jr(a−) blood type.
(a) Diagram showing the positions of ABCG2 null mutations identified in this study (see Table 1 for details). Blue boxes represent exons and broken lines introns. Red diamonds at the top represent the number of unrelated Jr(a−) subjects found with each mutation.
(b) Western blot analysis of ABCG2 expression in the membrane of RBCs from Jr(a+) and Jr(a−) individuals. Membranes were prepared from RBCs of 2 Jr(a+) (lanes 1 and 2) and 6 Jr(a−) (lanes 3 to 8) individuals and analyzed by polyacrylamide gel electrophoresis under reducing conditions without heat denaturation, and western blot with anti-ABCG2 BXP21 (top panel); similar results were obtained with rat anti-ABCG2 BXP34 (data not shown). The membranes were reprobed with a rabbit antiserum against p55, a membrane palmitoylated scaffolding protein of the RBC membrane (bottom panel). Of note, the different electrophoretic mobilities detected for ABCG2 here and in Figure 1e depends on the heat denaturation of lysates prior to electrophoresis.
(c) Flow cytometry analysis of ABCG2 expression at the surface of native RBCs from Jr(a+) and Jr(a−) individuals. RBCs from a Jr(a+) subject (blue profile) or a Jr(a−) subject (red profile) were labeled with mouse anti-ABCG2 5D3 (see Supplementary Figure 5 for a comparative study of 5D3 and HMR0921 antibodies); the histogram profiles of Jr(a−) RBCs, labeled with and without 5D3, were perfectly superposable. Similar results were obtained with mouse anti-ABCG2 F9123 (data not shown).

The transporter ABCG2 is the carrier of the Jr^a blood group antigen.
(a) The monoclonal antibody HMR0921 shows a strict Jr^a specificity with human RBCs. RBCs from a Jr(a+) subject (blue profile) or a Jr(a−) subject (red profile) were labeled with HMR0921. Of note, the histogram profiles of Jr(a−) RBCs labeled with or without HMR0921 were superposable.
(b) Cat RBCs are highly reactive with anti-Jr^a HMR0921. Cat RBCs were labeled with HMR0921 (blue profiles) or without (grey profiles).
(c) The ortholog of human ABCG2 is immunoprecipitated by anti-Jr^a HMR0921 from cat RBCs. Lysates were prepared from membranes of cat RBCs labeled with or without HMR0921, and corresponding immune complexes (lanes 3 and 4) were analyzed by polyacrylamide gel electrophoresis under reducing conditions with heat denaturation, and silver staining. The identity of the different bands present in HMR0921 immune complex was determined by mass spectrometry. Molecular weight markers are shown in lane 1 and pure HMR0921 in lane 2.
(d) Exogenous expression of human ABCG2 in K-562 cells results in cell surface expression of the Jr^a antigen. Live K-562 cells stably transfected with an episomal expression construct of ABCG2 cDNA (blue profiles) or the corresponding empty vector (red profiles) were analyzed by flow cytometry with anti-Jr^a HMR0921 (right panel) or anti-ABCG2 5D3 (left panel).
(e) Anti-Jr^a HMR0921 is able to immunoprecipitate human ABCG2. Lysates (lane 1: 1/500^th, lane 2: 1/1,500^th and lane 3: 1/4,500^th) were prepared from ABCG2-expressing or control K-562 cells labeled with HMR0921, and corresponding immune complexes (lanes 4 and 5) were analyzed by polyacrylamide gel electrophoresis under reducing conditions with heat denaturation, and western blot with anti-ABCG2 BXP21 (top panel) or anti-actin C4 (bottom panel).