Peroxidase labelled rabbit anti mouse immunoglobulins

Cell lysis buffer (125 mM Tris pH 6.8, 2 % SDS, 5 % 2-beta mercaptoethanol, 5 % glycerol with protease inhibitors: Sigma P8340 and 1mM PMSF) was used for the extraction of cultured cells and tissue lysis buffer (50mM Tris pH 6.8, 1 % EDTA, 10 % SDS, 5 % beta mercaptoethanol, 10 % glycerol with protease inhibitors) was used for the extraction of tissue samples (250 mg/ml). Bromophenol blue was added to the samples which were then boiled and separated by SDS-PAGE using 4 to 12 % polyacrylamide gels (Ref: NW04125BOX; ThermoFisher Scientific) and then electroblotted onto nitrocellulose membranes (Protan BA85, Whatman). Non-specific sites were blocked with 5 % skimmed milk protein and the membranes then incubated with monoclonal antibody supernatants (diluted: 1/10, except MANNES1E: 1/50 and MANDRA1: 1/100), followed by washing and incubation with secondary antibody (peroxidase-labelled rabbit anti-mouse immunoglobulins; 1/1000, Dako, Denmark). Antibody reacting bands were detected with SuperSignal West Femto chemiluminescent reagent (Cat No: 34094; ThermoFisher Scientific) and visualized with a ChemiDoc Touch imaging system (BioRad Ltd.).

Cultured cells were extracted in 125 mM Tris pH 6.8, 2% SDS, 5% 2-beta mercaptoethanol, 5% glycerol with protease inhibitors (Sigma P8340 plus 1 mM PMSF). Tissue samples (250 mg/ml) were extracted in: 50 mM Tris pH 6.8, 1% EDTA, 10% SDS, 5% beta mercaptoethanol, 10% glycerol with protease inhibitors. After the addition of bromophenol blue and after boiling, samples were subjected to SDS-PAGE using 4 to 12% polyacrylamide gels and transferred to nitrocellulose membranes (Protan BA85, Whatman). After blocking non-specific sites with 5% skimmed milk protein, membranes were incubated with monoclonal antibody (1/50), followed by washing and incubation with peroxidase-labelled rabbit anti-mouse immunoglobulins (1/1000, Dako, Denmark). Antibody reacting bands were visualized with West Femto chemiluminescent detection system (Pierce, Thermo Scientific).