Immobilon p 0.45 μm pvdf membranes

Western blot analysis was used to evaluate the specificity of mAbs towards recombinant CPE (aa 26–319, wild type) and CPE domains PFD (aa 26-202; GST-PFD-t-mCherry) and RBD (aa 203-319). For SDS-PAGE, the antigens were mixed with 3 × Laemmli loading buffer (150 mM Tris-HCl, pH 6.8, 6% SDS, 30% (v/v) glycerol, 7.5% (v/v) β-mercaptoethanol, 0.25% (w/v) bromophenol blue) and heated for 10 min at 95 °C. After cooling on ice, samples were loaded onto 12% polyacrylamide gels and separated electrophoretically according to standard procedures [80 (link)]. Subsequently, gels were transferred to methanol activated Immobilon® P 0.45 μm PVDF membranes (Millipore, Schwalbach, Germany). Membranes were blocked at 4 °C overnight in 2% skimmed milk in PBS-T and incubated for 1 h at room temperature with 1 μg/mL of CPE-specific antibodies diluted in blocking buffer. After three washing steps, detection was performed by biotin-labeled goat anti-mouse IgG (Dianova, Hamburg, Germany) diluted 1:10,000 in blocking buffer for 30 min at room temperature. Membranes were developed using alkaline phosphatase-conjugated streptavidin (Avidix™-AP, Fisher Scientific, Bremen, Germany) and CDP-Star as substrate (Perkin Elmer, Waltham, MA, USA). Development was documented on a ChemiDoc imaging system (Bio Rad Laboratories, Hercules, CA, USA).

Cell pellets were lyzed in PBS + 1% Nonidet-P40 with protein inhibitor cocktail (Roche, Basel, Switzerland) for 15 min on ice. Nuclei were spun down at 16,000 g for 15 min at 4 °C, supernatant was used for Western blotting. Cell lysates and EV were denatured in SDS-sample buffer at 100 °C for 3 min, and separated using 12% SDS-PAGE gels, after which proteins were transferred onto Immobilon-P 0.45 μm PVDF membranes (Millipore, Cork, Ireland). After blocking for 1–2 h in blocking buffer (0.5% Cold Fish Skin Gelatin (Sigma-Aldrich, St. Louis, CA) in PBS + 0.05% Tween-20), blots were incubated overnight at 4 °C with primary antibodies [anti-mouse-CD9 (eBioscience, clone KMC8, 1: 1000), anti-mouse-CD63 (MBL, clone D263-3, 1:1000), anti-mouse–galectin-3 (eBioscience, clone M3/38, 1:500), anti-MHCII-p55 (GenScript, Piscataway, NJ, custom Ab raised against MHCII bèta chain peptide sequence RSQKGPRGPPPAGLLQC, 1:5000), or anti-mouse-beta-actin (ThermoScientific, polyclonal PA1-16889, 1:5000)] in blocking buffer, and washed and incubated for 1–2 h with HRP-coupled secondary antibodies (Dako, cat P0450 and P0448, 1:5000). ECL solution (ThermoScientific, SuperSignal West Dura Extended Duration Substrate, cat. 34075) was used for detection on a Chemidoc imager (Bio-Rad, Hercules, CA). Images were analyzed by the Image Lab software (Bio-Rad, Hercules, CA).