Dramatic differences in level of expression of normal and mutated RPS19. Lysates of Cos-7 cells transfected with different GFP fusion protein constructs including GFP alone (1), GFP-WT RPS19 (2), GFP-mutated RPS19-Arg56Gln (3), GFP-mutated RPS19-Arg62Trp (4), GFP-mutated RPS19-Val15Phe+Thr55Met (5), GFP-mutated RPS19-Val15Phe (6), GFP-mutated RPS19-Thr55Met (7), and GFP-mutated RPS19-Gly127Gln (8) were analyzed by immunoblotting. We probed the immunoblots with chicken polyclonal anti-RPS19 antibody (panel A) or mouse monoclonal antichicken actin antibody (panel B). RPS19 antibody did not cross-react with GFP (panel A, lane 1), confirming its specificity. In contrast, the antibody detected GFP-WT and mutated RPS19 fusion proteins (panel A, lanes 2-8). The Val15Phe and Gly127Gln mutations, which impaired RPS19 nucleolar import, were associated with marked decrease in RPS19 expression (panel A, lanes 5, 6, 8) compared with normal RPS19 (panel A, lane 2). In contrast, expression of RPS19 with mutations located in the hot spot of mutations found in a large number of DBA patients: Arg56Gln, Arg62Trp (panel A, lanes 3-4, respectively) was the same as normal RPS19. Expression level of endogenous β-actin detected in a duplicate blot was similar in all samples, confirming that the amount of proteins loaded in each lane was comparable (panel B, lanes 1-8). Molecular weight standards (MWs) are shown on the left side of the blots.