Journal: Nature cell biology

Article Title: LIN28 phosphorylation by MAPK/ERK couples signaling to the post-transcriptional control of pluripotency

doi: 10.1038/ncb3453

Figure Lengend Snippet: (a) Schematic of the LIN28A domain structure with indicated phosphorylation sites, as mapped by mass spectrometry. Respective motifs and homologous sequences across several mammalian species are shown below each site. CSD = cold-shock domain; NLS = nuclear localization signal; CCHC = zinc finger domains. (b) Representative phosphopeptide MS/MS spectrum for S200. (c) Western blot analysis of LIN28A (S200) phosphorylation in HeLa cells stably expressing wild-type (WT) or phospho-null (S200A) FLAG-LIN28A. A representative image of three independent experiments is shown. (d) Western blot analysis of LIN28A (S200) phosphorylation in PA1 hECCs after 60-min treatment with a panel of inhibitors of proline-directed kinases. Tn1 = Torin1 (100 nM); PD = PD0325901 (1 µM); SB = SB203580 (2 µM); SP = SP600125 (20 µM); Ken = Kenpaullone (5 µM). Quantification of Western blot data is shown on top. n=3 independent experiments. Error bars represent s.e.m. *P < 0.05 (two-tailed Student’s t -test). (e) Western blot analysis of LIN28A (S200) phosphorylation in PA1 cells after 30-min pre-treatment with DMSO, PD0325901 (1 µM), or SB203580 (2 µM), followed by 30-min treatment with DMSO or PMA (200 nM). Cells were serum-starved for 16–20 h prior to addition of inhibitors. A representative image of two independent experiments is shown. (f) Western blot analysis of LIN28A (S200) phosphorylation in 293T cells stably expressing wild-type FLAG-LIN28A after transfection with RFP, wild-type (WT), constitutively active (R4F), or kinase-dead (K97M) MEK1. Cells were transfected, incubated for 48 h, and then serum-starved for 16–20 h prior to analysis. A representative image of two independent experiments is shown. (g) Western blot analysis of LIN28A (S200) phosphorylation in PA1 cells after 30-min stimulation with serum (10%), fibroblast growth factor (FGF) (100 ng/ul), or epidermal growth factor (EGF) (100 ng/ul). Cells were serum-starved for 16–20 h prior to stimulation. A representative image of two independent experiments is shown. Statistics source data are shown in Supplementary Table 4 . Unprocessed scans of blots are shown in Supplementary Fig. 5 .

Article Snippet: Drugs used were: Calyculin A (Cell Signaling), Torin1 (Tocris), PD0325901 (Stemgent), SB203580 (Invivogen), SP600125 (Sigma), Kenpaullone (Sigma), LY294002 (Cell Signaling), bFGF (Gemini), EGF (Peprotech), PMA (Cell Signaling), CHX (Sigma), and Doxycycline (Sigma).

Techniques: Phospho-proteomics, Mass Spectrometry, Tandem Mass Spectroscopy, Western Blot, Stable Transfection, Expressing, Two Tailed Test, Transfection, Incubation