p19^INK4dknockdown decreased GATA1 protein expression and ectopic GATA1 expression rescues the differentiation delay. (A) Representative images of western blotting analysis showing GATA1, 4.1R, tropomodulin (Tmod), HBG, p18^INK4c, KLF1, and CD44 expression in erythroblasts infected with Lucif shRNA or p19^INK4d shRNA (day 11 cells) (left). Quantitative analysis of protein expression data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein expression. (B) qRT-PCR analysis of GATA1 mRNA levels in erythroblasts infected with Lucif shRNA or p19^INK4d shRNA. The results were normalized to GAPDH mRNA. (C) Representative data of flow cytometry analysis of erythroblasts infected with Lucif shRNA, p19^INK4d shRNA, and p19^INK4d shRNA combined with GATA1 overexpression. GPA expression was monitored on D 9 cells (left) and expression of band 3 and α4-integrin on D 15 cells (right). The red numbers indicate statistical analysis of GPA-positive rate from 3 independent experiments. (D) Representative images of western blotting showing GATA1 levels in whole cell lysates prepared from erythroblasts (left). Quantitative analysis of expression data from 3 independent experiments (right). GAPDH was used as a loading control. Statistical analysis of data from 3 independent experiments and bar plot represents mean ± SD of triplicate samples. *P < .05, **P < .01, ***P < .001.