Fig. 4. Downregulation of B-Myb by RNA interference. HEL cells treated with 10^–8 M TPA for 16 hours were subjected to three rounds of transfection with 50 nM of the different FITC-conjugated siRNAs for three hours at one-day intervals. (A) 80 hours after TPA treatment, transfection efficiency was determined by flow cytometry to detect FITC-positive cells. The histograms indicate green fluorescence (FL1) on the horizontal logarithmic axis. The controls are mock-transfected cells. (B) 40 µg of total protein extract from siRNA-treated cells was used for a western blot analysis of B-Myb protein levels. The upper panel shows the signal obtained after probing with anti-B-Myb, whereas the lower panel is an image of the Ponceau Red staining of the same region of the filter after transfer. (C) RT-PCR analysis of gene expression following siRNA knockdown of B-Myb. cDNA was prepared from 1 µg RNA extracted from cells treated as described in A. Semi-quantitative RT-PCR was performed for the indicated genes, sampling reactions at 22, 25 and 28 cycles, using HPRT to normalise the cDNA input.