Rh dependency on RhAG is established early in erythropoiesis. (A) Flow cytometry histograms showing knockdown of RhAG and effect on band 3 and Rh. As described in “Small interfering RNA knockdown,” 1.5 × 10⁶ expanding erythroblasts were transfected with control siRNA or pooled siRNAs directed against RhAG. Cells were reseeded in differentiation medium RhAG (LA1818), and Rh (BRIC69) surface expression levels were monitored after 24 and 72 hours of differentiation and band 3 after 72 hours. (B) Quantification of RhAG and Rh knockdown at 24 and 72 hours in differentiation as shown in panel A, normalized against cells transfected with nontargeting siRNA. ★ indicates statistical significance (Student t test).