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Fig. 2
Figure 2. ISL1 phosphorylation by the BMP4-p38 MAPK signaling axis affects its protein stability (A and B) Luciferase assay of NIH3T3 cells expressing Isl1 and a Mef2c anterior heart field (AHF) enhancer reporter construct (pGL4-Mef2cPromoter-Luc2-AHF), treated either with DMSO or the p38 inhibitor SB202190 (n = 3) (A) and WB analysis for ISL1 of protein extracts used in the reporter assays (B). (C–F) Luciferase assay of NIH3T3 cells stably expressing Isl1 transfected with the Mef2c reporter construct and treated with or without 10% fetal bovine serum for 1 h (n = 3) (C) or with or without 0.01 μM anisomycin for 1 h (n = 3) (E), and WB analysis for ISL1 of protein extracts used in the reporter assays (D, F). (G) HA-tagged ubiquitin and FLAG-tagged ISL1 were transiently expressed in HEK293T cells. Cells were either treated with DMSO or SB202190 together with MG132 for 6 h before harvesting. Equivalent amounts of total protein were immunoprecipitated with anti-FLAG (M2) antibody and detected with an anti-HA antibody. (H) WB analysis for ISL1 of protein extracts of ISL1 overexpressing NIH3T3 cells treated with DMSO or SB202190 with or without the proteasome inhibitor MG132 for 16 h. (I) Scheme depicting the distinct stages of directed cardiac differentiation (top). MES, mesoderm; CPCs, cardiac progenitor cells; CMs, cardiomyocytes. WB analysis for ISL1 and activated phospho-p38 at different stages of directed differentiation of mESCs into CMs (middle). AP treatment of extracts from CPCs (bottom), showing that ISL1 is exclusively phosphorylated. (J) Heatmap representation of expression data for genes involved in BMP, activin, and FGF signaling during the course of cardiac differentiation (Wamstad et al., 2012). (K) Isl1 mRNA expression in mESCs overexpressing CMV-driven Isl1 treated with BMP4, basic FGF (bFGF), or activin for 30 or 60 min (top panel). WB analysis for ISL1 and activated phospho-p38 MAPK of protein extracts from Isl1−/− mESCs overexpressing CMV-driven Isl1 treated with BMP4, bFGF, or activin for 30 or 60 min (bottom panel).