Figure 8.

hnRNP E1 silenced cells reconstituted with WT, phosphomutant S43A or phosphomimetic S43E forms of hnRNP E1. (A) hnRNP E1-deficient NMuMG cells (NMuMG-/-hnRNP E1) were stably reconstituted with WT, S43A, S43E forms of hnRNP E1 and stable clones were treated −/+ TGFβ for 24 h. Cellular lysates were prepared and immunoprecipitated with α-hnRNP E1 (upper panel) or α-Flag (lower panel) to precipitate protein–mRNA complexes and RT/PCR was used to amplify mRNAs for EGFR and FAM3C. The data presented are typical of three independent experiments demonstrating similar binding trends (data not shown). (B) Immunoblot analysis confirming sh-RNA-mediated knockdown of hnRNP E1 in NMuMG-/-hnRNP E1 cells and reconstituted expression of wild-type (WT) and S43A (phosphomutant) and S43E (phosphomimetic) forms of Flag-tagged hnRNP E1. Blots depict levels of endogenous or overexpressed forms of hnRNP E1 using either α-hnRNP E1 or α-Flag immunoblotting. (C) Phase contrast images of unstimulated and TGFβ-treated (24 h) cells examining morphological changes after stimulation with TGFβ.