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. Author manuscript; available in PMC: 2014 Feb 11.
Published in final edited form as: Cancer Cell. 2013 Jan 31;23(2):171–185. doi: 10.1016/j.ccr.2012.12.021

Figure 6. Proteomics Analysis Demonstrate that the EC-secreted Soluble Form of Jagged-1 is C-terminally Truncated by ADAM17.

Figure 6

(A) Concentrated LPEC CM was fractionated by FPLC gel filtration. The fractions were applied to xhCRC cells containing the Hes-1 promoter - luciferase construct, and Hes-1 promoter activity was assessed (upper panel). Western blot detection of soluble Jagged-1 in combined adjacent fractions is shown in the lower panel.

(B) Jagged-1 from LPEC CM was immunoprecipitated using an N-terminal antibody and subjected them to deglycosylation followed by digestion. Mass spectrometric analysis of Jagged-1 proteins was performed with multiple peptides that identified consistent with the N-terminal region of Jagged-1 with a C-terminus at amino acid 1054.

(C) Mass spectrometric analysis of the ADAM17 cleavage site of Jagged-1. A synthetic peptide corresponding to aa1047–1061 of Jagged-1 was incubated with buffer only (left panel). ADAM17 (middle panel), or ADAM17 with its inhibitor TAPI-2 (right panel), and the reaction products were subjected to mass spectrometry. Peak 1 represents the intact substrate SLIAAVAEVRVQRRP, and Peak 2 represents the ADAM17 cleavage product, a 906.56 Da peptide that was determined to be VRVQRRP.

See also S5.