Pedigrees of two families carrying different ABCG2 premature stop mutations – co-segregation of the heterozygous mutation with reduced erythrocyte ABCG2 expression levels.

<p>Blood samples obtained from the 14 family members of the two healthy volunteer probands, carrying the premature stop mutations (see Fig. 3 -indicated with arrowheads) were analyzed for ABCG2 expression and the respective mutations. The RBC-G2 factor values, reflecting ABCG2 expression in erythrocytes, are shown in parentheses. Family members not available for blood donation are labeled by N.A.</p

Quantitative determination of ABCG2 expression in the erythrocyte membrane by flow cytometry.

<p>Anticoagulated blood samples of healthy volunteers were fixed in paraformaldehyde, stained with monoclonal antibodies recognizing human ABCG2, and subjected to flow cytometry (see Online Methods). Antibody staining was performed by BXP34 (Panel B), BXP21 (Panel C) and 5D3 (Panel D) mAbs specific for ABCG2, or the respective IgG control antibodies, followed by staining with PE-labeled secondary antibodies. In Panel E cells were stained with a FITC-conjugated anti-Glycophorin A mAb. Intact erythrocytes and erythrocyte ghost were gated based on the forward scatter (FSC) and side scatter (SSC) parameters (Panel A).</p

ABCG2 is differentially expressed in the red blood cells of individuals carrying homozygous wild-type, heterozygous polymorphic or premature stop codon mutant ABCG2 alleles.

<p>Boxplot presentation showing the median and the 25–75^th percentiles, whiskers represent 10–90^th percentiles. ABCG2 expression is calculated based on the combined reactivity of anti-ABCG2 mAbs (RBC-G2 factor – see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048423#s2" target="_blank">Methods</a>). Labels: individuals carrying wild-type ABCG2 (WT), polymorphic (Q141K, V12M) ABCG2 alleles, or a heterozygous stop mutation (STOP).</p

Detection of ABCG2 expression level by specific monoclonal antibodies.

<p>The Figure shows the correlation of the ABCG2 expression level detected by the 5D3 monoclonal antibody in the fixed whole cells, recognizing the ABCG2 protein on an extracellular epitope, and the weighed average of BXP21 and BXP34 antibodies (BXP34/3+BXP21)/2, recognizing intracellular epitopes of ABCG2. The correlation is linear; the value of the correlation coefficient R is 0.859.</p