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. 2014 Aug;34(15):2822–2832. doi: 10.1128/MCB.00206-14

FIG 4.

FIG 4

Ligand-dependent NOTCH2 and NOTCH3 signaling requires Adam10. (A) Immunoblots on cell lysates of monotypic coculture experiments with Adam10/17dKO-JAGGED2 cells, reconstituted with either Adam10 or Adam17 treated with DMSO or GSI. Blots were probed with antibodies for Notch2, N1ICD (Val1744), Myc (JAGGED2), Adam10, or Adam17. Endogenous N2EXT can accumulate only in the presence of Adam10 and GSI. Endogenous activated Notch1 (Val1744) could be detected only in cultures of cells expressing Adam10 and is inhibited by GSI. Overexpression of Adam17 did not lead to proteolysis of either endogenous Notch1 or Notch2. Molecular masses are indicated. (B) Relative Hes1 mRNA expression in Adam10/17dKO-JAGGED2 cells, reconstituted with either Adam10 or Adam17, subjected to monotypic coculture experiments in the absence or presence of GSI. Hes1 mRNA expression was induced only in the presence of Adam10 and could be completely blocked by GSI treatment. Experiments were performed in triplicate. Error bars represent means and standard deviations. (C) Coculture experiment of NOTCH2-HA-expressing Adam10/17dKO cells, reconstituted with either Adam10 or Adam17 with J1-expressing cells compared to parental OP9 cells. Panels display HA immunoblots of cell lysates as indicated. Diminished levels of NOTCH2 TMIC indicative of J1-dependent NOTCH2 processing were found in all cell types in the absence or presence of GSI. Upon activation, N2EXT fragments accumulated in cells expressing Adam10 by the addition of GSI (middle) and not in the presence of Adam17 (bottom). (D) Coculture experiment of NOTCH3-HA-expressing Adam10/17dKO cells, reconstituted with either Adam10 or Adam17 with Dll1-expressing cells compared to parental OP9 cells. Diminished levels of NOTCH3 TMIC are shown by HA immunoblotting upon activation. Genuine N3EXT accumulation was observed only after GSI treatment in the presence of Adam10 (middle) and not in the presence of Adam17 (bottom). (E) Immunoblotting of Adam10 and Adam17 endogenous proteins in Adam10 or Adam17 single knockout cells with OP9 cells as a positive control. (F) Immunoblotting for endogenous Notch2 in Adam10 or Adam17 single knockout cells cultured on coated Dll4-Fc molecules shows the formation of N2EXT to be Adam10 dependent, as N2EXT formation could not be observed in Adam10 knockout cells. In Adam17 knockout cells, treatment with GSI leads to the formation and accumulation of an N2EXT fragment. Molecular masses are indicated.