Fig 1: BCNU does not alter the activities of secretases. A) Partially purified β- and γ-secretase enzyme complexes from mouse brain homogenates were used to detect the activity of secretases using specific fluorogenic substrates. BCNU up to 40 μM did not inhibit the activities of either β- or γ-secretases. B) The activities of both ADAM17 and ADAM10 (α-secretase) were also not affected by BCNU at any of the concentrations tested. BCNU, 1, 3 bis (2-chloroethyl)-1-nitrosourea.
Fig 2: Dot blot reactivity of IgG from patient sera on recombinant ADAM10 moleculesThe reactivity of the IgG fraction purified from the sera of 6 Crc patients positive for the presence of auto-Abs anti-ADAM10 (C2, C27, C20, C9, C14, C19) and 6 Crc patients considered negative (C32, C50, C25, C11, C13, C45) was tested on human-recombinant ADAM10 (mature form lacking the pro-domain), mouse-recombinant ADAM10 (immature form including the pro-domain), ADAM10 pro-domain synthetic peptide (32 aa within the pro-domain sequence), human-recombinant ADAM17 (immature form including the pro-domain) and purified human ceruloplasmin (Cp) proteins spotted (200 ng/spot) onto nitrocellulose membrane. The reactivity of the anti-ADAM10 ectodomain Ab that recognizes both mature and immature ADAM10, anti-ADAM10 pro-domain Ab, anti-ADAM17 and anti-ceruloplasmin Abs were used as controls.
Fig 3: Expression of ADAM10 in primary tumorImmunohistochemistry analysis of ADAM10 expression in normal intestinal epithelia tissue a. and tumoral tissue b.-f.. Arrows indicated in panel a. the few ADAM10 positive monocytes resident in the cryptae, and in panels a.-f. the stronger reactivity of tumor cells invading the stroma.
Fig 4: Co-localization of immunofluorescence signals from anti-ADAM10 pro-domain Ab and IgG from anti-ADAM10 positive patient serumDouble staining was performed with rabbit anti-ADAM10 pro-domain (ab39178, Abcam), and either IgGs purified from Crc patient serum or mouse anti-HLA class I (Santa-Cruz Biotechnology). Secondary Abs were Alexa-546-conjugated goat-anti-rabbit IgG and Alexa-488-conjugated rabbit-anti-mouse IgG or FITC-conjugated goat-anti-human IgG. Cell nuclei were stained with Hoechst-33342, and differential interference contrast (DIC) images were also acquired. Staining was assessed by immunofluorescence confocal microscopy (Leica TCS SP5 Laser Scanning Confocal) and images acquired with LAS-AF (Leica) software; magnification 63X. Images from single channel and merged images are shown. Images were linearly adjusted for brightness and contrast using ImageJ 1.47v software.
Fig 5: ADAM10 isoforms expression in Crc specimensA. Anti-ADAM10 WB analysis in Crc specimens from representative patients with (Ab-pos, C27, C38, C9, C21, C36) or without (Ab-neg, C32, C52) serological reactivity against ADAM10. Anti-β-actin reactivity was used for protein-loading control and OD normalization. B. Quantitative analysis of WB reactivity detected on Ab-pos (n = 15) and Ab-neg (n = 12) patient specimens (mean +/− SEM 3 experiments; student's t-test; ** p < 0.01; *** p < 0.001). The ratio between pro-protein and mature ADAM10 signals is reported.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Human ADAM10 Protein, CF