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. Author manuscript; available in PMC: 2010 Sep 1.
Published in final edited form as: Nat Cell Biol. 2010 Feb 14;12(3):286–293. doi: 10.1038/ncb2029

Figure 5.

Figure 5

Modulation of hnRNP E1 expression or its posttranslational modification alters translation of Dab2 and ILEI and sensitivity of NMuMG cells to TGFβ-induced EMT. (a) Phase contrast images of unstimulated and TGFβ-treated (24 hr) WT, E23 and E2KD cells examining morphological changes post TGFβ-stimulation. Images were taken at 10× magnification. (b) IB analysis monitoring Dab2, ILEI, N-cadherin, vimentin and b-actin protein levels in WT, E23 and E2KD cells treated with TGFβ for the times indicated. (c) In vivo validation of Ser43 as the hnRNP E1 phosphorylation site. WCLs derived from NMuMG, KIM2 and KIWT6 cells were immunoprecipitated with α-hnRNP E1 antibody and analyzed by IB with α-phospho serine antibody (top panel) and α-hnRNP E1 antibody (second panel). TGFβ-dependent Akt activation analyzed by IB analysis of WCLs derived from NMuMG, KIWT6 and KIM2 cells treated with TGFβ for the times indicated (third and bottom panel). (d) IB analysis examining Dab2, ILEI, N-cadherin, vimentin and β-actin protein levels in cells treated with TGFβ for the times indicated. (e) & (f) IVT and RNA pull-down assays with cytosolic extracts from SH14, KIWT6 and KIM2 cells treated with TGFβ for the times indicated to examine translational silencing of chimeric Luc-Dab2/BAT cRNA (d) and temporal association of the modified hnRNP E1 with the Dab2/BAT cRNA (e). (g) Role of hnRNP E1 on EMT is mediated by Dab2 and ILEI. IB analysis of WCLs derived from SH14 cells, un-transfected or transiently transfected with ILEI, Dab2 or control-A siRNA to confirm knockdown of Dab2 and ILEI, respectively (first and second panel, respectively). IB analysis examining N-cadherin, vimentin and Hsp90 protein levels in these cells (third, fourth and bottom panel, respectively). Full scans of (b), (c), (d), (e), (f) and (g) are shown in SI Fig. S7.