Sodium dodecyl sulfate polyacrylamide gel

After 24 h of coculture, total proteins were extracted from islets using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing protease and phosphatase inhibitor. Homogenates were purified by centrifugation at 12,000 × g at 4°C for 12 min, and protein concentrations were determined using a bicinchoninic acid protein assay (Thermo Fisher Scientific). Equal amounts of protein extract were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 12% Tris-HCl gels (Bio-Rad Laboratories Inc, Hercules, CA, USA). The separated proteins were then transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories Inc) and incubated for 1 h with the following primary antibodies: anti-signal transducer and activator of transcription (STAT) 3 (Cell Signaling Technology, Danvers, MA, USA), anti-phosphorylated STAT3 (pSTAT3) (Cell Signaling Technology), and anti-β actin (Sigma-Aldrich). Next, the membranes were incubated with horseradish peroxidase–linked anti-rabbit IgG (GE Healthcare, Buckinghamshire, UK) at room temperature for 1 h. The antigen–antibody complex was detected using the ECL Prime Western Blotting Detection kit (GE Healthcare).

Western blot analysis to detect B. bovis-specific antibodies to RAP-1 in bovine sera was performed using recombinant RAP-1 (rRAP-1), according to the previously described method [13 (link)] with some modifications. Briefly, rRAP-1 was boiled for 3 min in sample buffer (DGel Sciences, Montreal, Canada) and separated by electrophoresis using sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad, Hercules, CA, USA). Transfer to nitrocellulose was performed using standard techniques [24 (link)] and membranes were blocked in tris-tween-20 buffer containing 5% skim milk. Bovine serum antibody bound to the rRAP-1 band was detected with HRP-conjugated goat anti-bovine IgG (KPL, Gaithersburg, MD, USA) [15 (link), 17 (link)].

Cells were lysed in 2× Laemmli Sample Buffer (Bio-Rad, 1610737) supplemented with 5% β-mercaptoethanol, and 1× Halt Phosphatase Inhibitor Cocktail (ThermoFisher, 78426) and boiled for 10 min. Protein quantification was completed using BCA assay and 30 µg of protein was added per lane to a 10% or 4–20% sodium dodecyl sulfate polyacrylamide gel (Bio-Rad) and transferred to a polyvinylidene difluoride membrane (Millipore Sigma, IPVH00010). Membranes were then blocked for 1 h at room temperature in Intercept Blocking Buffer (LI-COR, 927-60001). Membranes were incubated overnight at 4 °C with primary antibody and subsequently incubated with secondary antibodies for 1 h at room temperature. Band visualization was completed using the LI-COR Odysessy CLx system.

Culture media were obtained from BD Difco (Sparks, MD, USA) and JT Baker (Phillipsburg, NJ, USA). All other chemicals, solvents and substrates used were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA) and JT Baker. Kits for expression, DNA isolation and purification, as well as polymerase enzyme and Ni–NTA resin were obtained from Qiagen (Valencia, CA, USA). Restriction enzymes and DNA ladder were purchased from New England Biolabs (Beverly, MA, USA). pJET1.2/blunt vector was purchased from Fermentas (St Leon-Rot, Germany). The chemicals, including the protein molecular weight markers used in the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and agarose gels analysis and the Econo gradient pump system were purchased from BioRad (Hercules, CA, USA). Lysozyme was obtained from Research Organics (Cleveland, OH, USA) and bovine serum albumin (BSA) was purchased from Pierce (Rockford, IL, USA). Finally, for thin-layer chromatography of silica gel was purchased from Merck KGaA (Darmstadt, Germany).

Cells were incubated with M-COPA for 24 h, and then lysed as described previously [51 (link)]. Proteins in cell lysates were separated by 4–15% sodium dodecyl sulfate–polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) electrophoresis, followed by electroblotting onto a nitrocellulose membrane (Bio-Rad Laboratories). Primary and secondary antibodies used for immunostaining are listed in the Supplementary Table 1. Immunoreactive bands were visualized and quantitated by using the ODYSSEY^® CLx Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).