(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.