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Figure 1 (A) DMS53 cells were treated with 5 nM BMP-2 for 6 or 12 h. Total extracts were obtained and analyzed by immunoblotting for the neural markers indicated. (B) DMS53 cells were treated for 4 h with the indicated amounts of BMP-2 or TGF- (upper panel) or with 10 nM BMP-2 for the times indicated (lower panel). TH and actin expressions were analyzed by immunoblotting and/or semiquantitative RT–PCR. (C) C17.2 cells were cotransfected with a TH promoter-driven reporter construct and the combinations of Mash1 and/or E47 expression vectors indicated. Luciferase assay was performed after treatment for 16 h with 5 nM BMP-2 (filled bars) or no addition (empty bars). The results are expressed as mean s.e.m. of triplicates from four independent transfections. (D) Northern blot analysis was performed with total RNA from DMS53 cells treated for the indicated times with 10 nM BMP-2 as described in Materials and methods.
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 | Figure 2 (A) Cell extracts were obtained from DMS53 cells treated with 5 nM BMP-2 for the times indicated and analyzed by immunoblotting. (B) Quantifications of Mash1 (filled circles) and Id1 (filled squares) protein levels from five separate assays were performed using  -tubulin as normalization and were expressed as fold-change over control samples at time zero and expressed as means.e.m. (C) DMS53 cells were treated for 2 h with the indicated amounts of BMP-2 and Mash1 expression was analyzed by immunoblotting. (D) Parental and two independent myc-Mash1 overexpressing clones were treated for 2 h with 5 nM BMP-2 and expressions of ectopic Mash1 (upper panel) or endogenous Mash1 (lower panel) were analyzed by immunoblotting.
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Figure 3 (A) DMS53 cells were treated with 50  M LLnL and/or 5 nM BMP-2 for 2 h and expression of Mash1 was analyzed by immunoblotting. (B) Extracts from HEK-293 cells transfected with the indicated expression constructs and treated overnight with 50 M LLnL were subjected to immunoblotting to detect myc-Mash1 expression before (left panel) or after Ni2+-NTA-agarose purification (right panel).
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 | Figure 4 (A, B) Extracts from HEK-293 cells transfected with the expression constructs indicated, treated or not with 50 M LLnL for 8 h, were analyzed by immunoblotting. (C) HEK-293 cells, transfected with the constructs indicated, were pulsed with [35S]methionine for 3 h and labeled proteins were chased in unlabeled media for different times. Labeled Mash1 was immunoprecipitated and visualized by SDS–PAGE. Quantification of Mash1 levels from three to four separate assays is expressed as means.e.m. of percentage values of samples at time zero.
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Figure 5 (A) Immunoblot analysis of tetracycline (Tet) responsiveness of several stable DMS53-Id1 clones. (B) Id1-overexpressing clones (Id16 and Id17) were incubated in the presence of 30 ng/ml of tetracycline overnight, and then incubated for 8 h in the presence of different concentrations of tetracycline. Expressions of Mash1 and Id1 were analyzed by immunoblotting. (C) Parental and two Id1-overexpressing clones (Id16 and Id17) were incubated overnight with 30 ng/ml of tetracycline in order to block ectopic Id1 expression. Then cells were deprived of tetracycline for the times indicated and expressions of Mash1 and Id1 were analyzed by immunoblotting.
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 | Figure 6 (A) E47 expression of DMS53 cell clones, stably overexpressing E47, was analyzed by immunoblotting and is shown in the upper panel. Parental and two independent clones (E10 and E17) were treated for 2 or 4 h with 5 nM BMP-2 and expressions of endogenous Mash1 and Id1 were analyzed by immunoblotting (lower panel). (B) Wild-type or Id1-/- MEFs were transfected with the indicated expression constructs. After 24 h, cells were splitted and treated with 10 nM BMP-2 for 3 h. Expressions of Mash1, Id1 and tubulin were analyzed by immunoblotting.
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Figure 7 HEK-293 cells, transfected with the constructs indicated, were labeled with [35S]methionine or 32Pi for 3 h as described in Materials and methods. Labeled Mash1 or E47 was immunoprecipitated and visualized by SDS–PAGE (see text).
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 | Figure 8 (A) HEK-293 cells, transfected with the wild-type or mutant Mash1 constructs, with or without E47 expression construct as indicated, were labeled with [35S]methionine and Mash1 was immunoprecipitated as above. (B) C17.2 cells were cotransfected with a TH promoter-driven reporter construct and the indicated combinations of wild-type and mutant Mash1 with (filled bars) or without (empty bars) E47 expression vectors. The results are expressed as means.e.m. of triplicates from five independent transfections. (C, D) Pulse-chase assays and quantification were performed in HEK-293 cells, transfected with the constructs indicated, as described in Figure 4B.
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Figure 9 (A, B) HEK-293 cells, transfected with the wild-type Mash1 with or without E47 expression constructs as indicated, were labeled with [35S]methionine and treated for 2 h with the drugs indicated (FK (forskolin); H89; DRB (5,6-dichloro-1--ribofuranosylbenzimidazole); U (U0126); bisI (bisindoleylmaleimide I); SB (SB203580); emo (emodin); api (apigenin)). Immunoprecipitated Mash1 and co-immunoprecipitated E47 were visualized as described above. (C) HEK-293 cells were transfected with the indicated expression constructs and siRNA for CK2 subunit mRNA or an unrelated siRNA directed against uPF2K as a control. After 24 h, cells were either metabolically labeled and both Mash1 and co-immunoprecipitated E47 were visualized as described above, or lysed and both CK2 and tubulin were analyzed by immunoblotting. (D) DMS53 cells were treated with DRB, emodin or apigenin for 2 h and Mash1 or -tubulin were visualized by immunoblotting. (E) Immunoprecipitates of extracts from HEK-293 cells transfected with the indicated constructs were split and either incubated with recombinant CK2 and [ -32P]ATP (upper panel) or subjected to immunoblotting in order to visualize the relative amounts of immunoprecipitated Mash1 (lower panel).
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 | Figure 10 Model of mechanisms of repression of Mash1 by BMP-2/Id1. (A) In differentiating neurogenic precursors, Mash1 heterodimerizes with E proteins, which promotes phosphorylation of Mash1 on Ser152, increasing further heterodimer interaction. (B) Activation of the BMP signaling pathway leads to increased levels of Id proteins. Id proteins sequester E proteins away from Mash1, which leads not only to transcriptionally inactive complexes but also enhances degradation of Mash1 monomers.
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