FIGURE 4.

hnRNPA2/B1 regulates Cbx3 gene expression. A and B, knockdown of hnRNPA2/B1 down-regulated Cbx3 gene expression. Cbx3-/Hnrnpa2/b1-specific siRNAs and random siRNA control were transfected into day 3 differentiating cells; after an additional 48 or 72 h of culture, total RNA and protein were harvested and subjected to RT-qPCR (A) and Western blot analysis (B). α-Tubulin was included as internal control. C and D, Hnrnpa2/b1 gene expression was not affected by Cbx3 overexpression. E and F, hnRNPA2/B1 overexpression significantly increased Cbx3 gene and protein expression levels. Undifferentiated ES cells were nucleofected using nucleofector II with different amounts of hnRNPA2/B1 expression plasmids pCMV5-Hnrnpa2/b1. Nucleofected cells were plated on dishes coated with 5 μg/ml collagen IV and cultured for 3–4 days in DM. Total RNA and protein were harvested and subjected to RT-qPCR (E) and Western blot analyses (F). Appropriate amounts of empty vector pCMV5 (control) were included to ensure total DNA concentrations for all experiments were equal. The data presented here are representative or mean ± S.E. of three independent experiments, *, p < 0.05. Numbers below the bands in B, D, and F indicate the average relative expression levels of target proteins from three independent experiments.