Effect of ARHGEF3 RNAi on Tf uptake in erythroid cells. Tf binding and uptake were assayed in K562 cells that have been transduced with either the lenti-GFP/RNAi-ARHGEF3 construct or lenti-GFP/mmRNAi-WDR66, which was used as a control. (A-B) In arhgef3-depleted K562 cells (B) Tf uptake was dramatically reduced compared with the control (A; white arrows indicate cells showing no Tf). (C) Tf-633 uptake by K562 control cells (n = 105) versus RNAi-ARHGEF3-transduced cells (n = 104), (**P < .01 in the graph). (D) Tf binding to the membrane is unaltered in ARHGEF3 knockdown cells. Control and ARHGEF3-depleted K562 cells were incubated with Tf-633 at 4°C, and excess of unbound Tf was removed by washing. Membrane-bound Tf was measured by flow cytometry, MFI is on the x-axis, and the number of cells assessed is on the y-axis.