Figure 3. Construction of a DNA-binding defective and siRNA-resistant B-Myb mutant. (A). Schematic illustration of the DNA-binding deficient and RNAi resistant B-Myb mutant. The nucleotide and amino acid sequences of the wt and mutant B-Myb at the B-myb_siRNA4 target region are shown. (B). Equal amounts of GST-B-Myb-wt and GST B-Myb-N174A proteins were subjected to an EMSA experiment using a Myb-binding site oligonucleotide (top). Aliquots of the protein extracts were analyzed by SDS-PAGE and stained with Coomassie brilliant blue to confirm equal protein concentration (bottom). (C). HepG2 cells were first transfected with expression vector for B-Myb (N174A) mutsiRNA_4. Equal aliquots of these cells were subsequently transfected with control siRNA, B-myb_siRNA3 or B-myb_siRNA4 and analyzed by western blotting 24 h later.