Fig. 2

Identification of α-secretase cleavage sites on CNTNAP2. a Peptide in vitro cleavage. Two peptides were incubated in the assay buffer with or without ADAM17 at 37 °C for 24 h and then were sent to Mass Spectrometry analysis. The extracted ion chromatogram showed two intact peptides (top) and cleaved complementary fragments (bottom). Peptide 1 (M108-G84) was cleaved by ADAM17 at 97H/L96. Peptide 2 (L96-N72) was cleaved at 80 A/79I. b N-terminal sequencing results identified the first 5 amino acids (aa) of CTFα1 as IRNGV and the first 5 aa of CTFα2 as LDSAS. The bottom sequence shows the last 108 to 72 aa of CNTNAP2 from the C-terminus and α-secretase cleavage sites at L96 and I79. c Protein ladders of the C-terminal CNTNAP2. C79 corresponded to the size of CTFα1, and C96 showed the same size as CTFα2. d, e Mutations at the α-secretase cleavage sites L96 and I79 affected CNTNAP2’s cleavage. Wildtype (WT) or mutant plasmids were co-transfected with empty vector (EV), ADAM10 (AD10), or ADAM17 (AD17) into HEK cells. Alterations in the migration rate were observed in constructs containing D98R. Two upper/lower blots show samples from the same experiment. The parallel blots were processed in the same electrophoresis chamber and scanned together simultaneously. n = 3 independent experiments, two-way ANOVA followed by Dunnett’s multiple comparisons test (compare ADAM10 and ADAM17 with Control for each plasmid), row factor = mutation, column factor = ADAM10/17 overexpression. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. All the results are expressed as mean ± SEM