Protan ba85

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).

Samples were mixed with SDS buffer (125 mM Tris pH 6.8; 2% SDS; 5% 2-beta mercaptoethanol; 5% glycerol; with bromophenol blue), boiled and subjected to SDS-PAGE using 10% polyacrylamide gels and transferred to nitrocellulose membranes (Protan BA85, Whatman). Non-specific sites were blocked with 5% skimmed milk protein, membranes washed with PBS and then incubated with primary monoclonal antibodies against: XIRP1, (Xin-alpha (D-8); sc-166,658; 1/100; Santa Cruz Biotechnology, Insight Biotechnology Ltd., Wembly, UK); or GST, mAb 17A10 (1/100). This was followed by washing in PBS and incubation with secondary antibody (peroxidase-labelled rabbit anti-mouse immunoglobulins, P0260; 1/1000; Dako, Denmark). Alternatively, for the detection of annexin A5, pAb ANXA5 (Abcam; ab14196; 1.4 μg/mL) primary antibody followed by goat anti-rabbit Ig HRP (P0448; Dako; 1/1000) secondary antibody. All antibodies were diluted in PBS containing 0.05% Triton X, 0.1% BSA, 1% horse serum and 1% fetal bovine serum. XIRP1 and annexin A5 antibody reacting bands were detected with SuperSignal West Femto chemiluminescent reagent (Cat No: 34094; ThermoFisher Scientific) and GST antibody reacting bands were detected with SuperSignal West Pico Plus chemiluminescent reagent (Cat No: 34580; ThermoFisher Scientific) and visualized with a ChemiDoc Touch imaging system (BioRad Ltd.).