Ab39153

Primary antibodies for PLA and western blotting are described in Table 1. Secondary antibodies (horseradish peroxidase (HRP)-Goat anti-mouse and HRP-Goat anti-rabbit) were purchased from Bio-Rad and GE Healthcare. Secondary antibodies conjugated to PLA probes were purchased from Sigma-Aldrich (Merck).Table 1

List of antibodies used for western blotting (WB) and proximity ligation assay (PLA)

Antibody name	Company	Dilution (WB)	Dilution (PLA)
Anti-ADAM10	Abcam (ab39153)	1:4000	1:4000
Anti-Amyloid beta precursor protein C-terminus [Y188]	Abcam (ab32136)	1:5000	1:5000
Anti-APP C-terminus [C1/6.1]	BioLegend (802801)	–	1:1000
Anti-BACE1	Cell Signaling Technology (D10E5)	1:1000	1:200
Anti-PSD-95	Abcam (ab2723)	–	1:20 000
Anti-PSD-95	Neuromab, k28/43 (75-028)	1:1000	–
Anti-Synaptophysin	Enzo (VAM-SV011F)	1:10 000	1:3000

Formalin-fixed, paraffin-embedded sections from available Crc specimens and normal mucosa underwent antigen retrieval and quenching with 3% hydrogen peroxide. Sections were incubated 12 hr at 4°C with anti-ADAM10 N-terminal antibodies (1:500 Sigma; 1:1000 ab39153, Abcam), followed by labelled Envision anti-rabbit System (Dako). The immunoreaction was revealed by HRP using 3,3′diaminobenzidine (Biogenex), the slides were slightly counterstained with Harris's hematoxylin, analysed with optical microscope Axioskope 2 (Zeiss) and images acquired with AxioVision 4.4 system (Zeiss). Available tumor specimens frozen at −80°C were mechanically broken and incubated with 1%TritonX-extraction buffer containing protease- and phosphatase-inhibitors (Sigma). Proteins were resolved by SDS-PAGE and WB analysis was performed with anti-ADAM10 (AB936, R&D-Systems) and anti-β-actin (Sigma) Abs. Secondary Abs were HRP-conjugated goat-anti-rabbit IgG and rabbit-anti-mouse IgG (Dako). The protein loading was normalized by ponceau-staining and β-actin immunostaining.

Human-recombinant ADAM10 (aa 214-672, lacking the pro-domain, R&D Systems 936-AD-020), mouse-recombinant ADAM10 (aa 19-673, including the pro-domain, Calbiochem PF124), ADAM10 pro-domain synthetic peptide (Abcam ab41165; 32 aa within the pro-domain, sequence not available because of property used as immunogen to generate the anti-ADAM10 pro-domain antibody), human-recombinant ADAM17 (aa 1-671, including the pro-domain, Calbiochem PF133) and human ceruloplasmin purified from plasma (Alexis Biochemicals, ALX-200-089) were spotted (200 ng each spot) onto nitrocellulose membrane and tested with the fraction of IgG purified from the sera of six Crc patients considered positive for the presence of auto-antibodies anti-ADAM10 and six patients considered negative. Membrane strips were incubated for 2 hr at 20°C with IgG purified from Crc patients sera (50 μg/ml of IgG working dilution) and reactivity revealed with HRP-conjugated anti-human-IgG (Southern Biotechnology) followed by ECL reaction and film exposure. As controls, the reactivity of the rabbit anti-ADAM10 Ab (Abcam, ab39153) that recognizes both mature and immature ADAM10, rabbit anti-ADAM10 pro-domain Ab (Abcam, ab39178), rabbit anti-ADAM17 (Abcam, ab39163) and sheep anti-ceruloplasmin (Abcam, ab8813) were tested on the spotted proteins. Immunoreactivities were revealed as described above.