F2 mutations at positions 20210 and 20221 cause an increased abundance of F2 mRNA and protein. (A) In the hybrid genes, the 3′UTR including the poly(A) signal (AATAAA), the CS, the U-rich region, and 62 nt's of the 3′FS of the HBB gene were replaced by the respective sequences of F2 with either the normal F2 WT or the F2 20210^*A, F2 20210^*C, or F2 20210^*T mutations at the CS (pos. 20210) or the F2 20221^*A, F2 20221^*G, or F2 20221^*T mutations 11 nt's 3′ of the CS (pos. 20221). (B) Northern blot with an HBB-specific cRNA probe of cytoplasmic RNA preparations from cells cotransfected with the indicated hybrid constructs and the WT+300 E3 control. The RNA loaded in the control lanes 9 and 10 originate from cells that were either transfected with the WT+300 E3 control plasmid only or were not transfected. The bar diagram shows the fold difference relative to the F2 WT mRNA expression levels ± SD after normalization for transfection efficiency. Bars 1 to 8 represent quantification of lanes 1 to 8. (C) Immunoblot of protein lysates from cells transfected with constructs as shown in panel B. The blot was probed with an HBB-specific antibody.