Polysome association of mRNAs in erythroblasts deficient for Rps19 and Rpl11. (A) Polysomal- and subpolysomal-derived cRNA generated from parental I/11 erythroblasts (wt), and cells transduced with Sc shRNA or Rps19 shRNA was hybridized to expression arrays. After normalization, arbitrary ratios of polyribosomal recruitment were calculated (see “DNA microarrays and data processing”). Transcripts that are differentially expressed between control cells (wild-type and Sc shRNA) and Rps19-deficient cells were identified by ANOVA (false discovery rate < 0.05) and hierarchically clustered. Three columns for each class represent independent biologic replicates. (B) Protein levels of Bag1 and Csde1 in I/11 erythroblasts 3 days after transduction with Sc and Rps19 shRNA. Actin and Erk1/2 served as loading controls. Quantified expression, corrected for actin, is indicated below the blots. (C) Protein levels of Rps19, Rpl11, Bag1, and Csde1 in primary erythroblasts obtained from E12.5 fetal liver cells 3 days after transduction with Sc, Rps19A, and Rpl11A shRNA. Actin served as a control. Numbers indicate blot quantifications corrected to actin. Asterisk indicates a nonspecific band in the Rps19 panel. (D) qRT-PCR on RNA obtained from sorted mouse BM fractions, day E13.5 mouse fetal liver (FL), and 5 days in vitro erythroid culture of the fetal liver cells (EB, erythroblasts). LSK indicates Lin⁻Sca1⁺c-Kit⁺; CMP, common myeloid progenitor (Lin⁻c-Kit⁺CD34⁺CD16/CD32^low); GMP, granulocyte-monocytic progenitor (Lin⁻c-Kit⁺CD34⁺CD16/CD32^high); MEP, megakaryocytic-erythroid progenitor (Lin⁻c-Kit⁺CD34⁻CD16/CD32^low). Expression is shown relative to LSK levels on a logarithmic scale. Error bars represent ± SEM (n = 4). (E) Mouse erythroblasts (I/11) were induced to differentiate. Every 12 hours, protein samples were prepared to be analyzed for expression of Bag1 and Csde1. Actin served as a loading control. The positions of size markers are indicated.