Bicinchoninic acid kit

Whole-cell lysates were prepared using a mammalian protein extraction reagent (Thermo Fisher Scientific), and protein concentrations were determined using a bicinchoninic acid kit (Thermo Fisher Scientific). Total protein (25 μg) in each cell lysate was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted using specific antibodies. Proteins were visualized using the Clarity™ Western ECL substrate (Bio-Rad, Hercules, CA, United States) and ImageQuant™ LAS 4000 mini (GE Healthcare, Piscataway, NJ, United States). Band intensities were measured using the ImageJ software (v1.53), and the relative expression value of each protein was calculated after normalization based on β-actin expression level. Antibodies against α-actinin (#6487), focal adhesion kinase (FAK) (#3285), talin-1 (#4021), and vinculin (#4650) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, United States), and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, United States).

Plasmids encoding the heavy chain and light chain of Abs were transiently co-transfected in pairs, at equivalent molar ratios, into cultured mammalian human embryonic kidney HEK293F cells in Freestyle 293F medium (Invitrogen, CA, USA, 12338018) following the standard protocol [34 (link),35 (link)]. Culture supernatants were collected after 6 days by centrifugation and filtration (0.22 μm, polyethersulfone; Corning). Abs were purified from the culture supernatants using a CaptivA™ Protein A-agarose chromatographic column (Repligen, MA, USA) and were extensively dialyzed to achieve the final composition of phosphate-buffered saline (PBS; pH 7.4). Likewise, the plasmid encoding the pMHC SCT protein was transfected into HEK293F cells. The pMHC protein was purified from the culture supernatant using Ni-NTA resin (GE Healthcare, IL, USA). Protein concentrations were determined using a bicinchoninic acid kit (Thermo Fisher Scientific, Waltham, MA, USA). To prepare an Ab-screening antigen, the purified pMHC SCT proteins were biotinylated using a BirA500 kit (Avidity LLC, Colorado, USA) following the manufacturer’s instructions [35 (link)].

Total protein was extracted from EC cells and tissues through phenylmethylsulfonyl fluoride and protease inhibitors according to the instructions. After the samples had been lysed at 4 °C for 15 min and centrifuged at 15,000 r/min for 15 min, a bicinchoninic acid kit (23227, Thermo Fisher Scientific Inc.) was used to provide an estimation of the protein concentration within the supernatant. After the protein had been separated by polyacrylamide gel electrophoresis methods, the protein on the gel was electroblotted to a polyvinylidene fluoride membrane and subsequently blocked by 5% bovine serum albumin (BSA) at room temperature for 1 h. The membrane was subsequently probed overnight at 4 °C with the diluted primary rabbit antibodies (Abcam Inc.) against p53 (0.5 μg/mL, ab183544), FOXP3 (10 μg/mL, ab215206) and GAPDH (0.5 μg/mL, ab181602). The membrane was then re-probed with horseradish peroxidase labeled goat anti-rabbit IgG secondary antibody (0.1 μg/mL, ab205718, Abcam Inc.) at room temperature for 1.5 h. Developing solution (NCI4106, Pierce, Rockford, IL, USA) was subsequently added to the membrane for color development purposes. Image J 1.48u software (Bio-Rad, Hercules, CA, USA) was employed for protein quantitative analysis, which was expressed as the ratio of the gray value of each protein to that of the internal reference.

M. paratuberculosis K10 and JTC-1285 cultures were centrifuged in pre-weighed 50 mL conical tubes at 3200 × g for 15 min at room temperature. The supernatant was filter sterilized with 0.22 μm polyethersulfone (PES) filter (Nalgene). Further, it was size fractionated by ultrafiltration (Corning Ultra spin columns, 5000 MWCO) and the filtered volume retained on the membrane was dialyzed twice against 10 mM phosphate buffered saline (PBS) (pH 7.2). The concentrated culture filtrate proteins were quantified using bicinchoninic acid kit (Pierce) and stored at −20 °C. To obtain the lysate, the bacterial cell pellet was resuspended in protein lysis buffer (100 mM Tris–Cl, 100 mM NaCl, 5 mM MgCl₂, 1 mM PMSF, complete ultra protease inhibitor cocktail, pH 7.5) and placed in microcentrifuge tubes containing 0.1-mm zirconia/glass beads. Tubes were shaken in the Mini Bead-beater cell disrupter for four 45 s pulses followed by 1-min incubation on ice. Cellular debris and beads were pelleted by centrifugation at 3200 × g for 20 min. The supernatant was quantified for protein using bicinchoninic acid kit (Thermo Fisher Scientific, Rockford, IL) and stored at −20 °C.

Protein was extracted using RIPA-reagent LS (Aidlab Biotechnologies, Beijing, China) according to the manufacturer’s protocols. Protein level was measured using a Bicinchoninic Acid kit (Thermo Scientific). Then, 40 μg of protein lysate was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), and then immunoblotted with antibodies against phospho-AMPK_α1 (Thr-172; 1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), AMPK (1:1000; Abcam, Cambridge, UK), PPARα (1:1000; Abcam), and GAPDH (1:2000; Abcam). Subsequently, they were incubated with horseradish peroxidase-conjugated secondary antibodies (MultiSciences (Lianke) Biotech, Hangzhou, China), visualized using an ECL Plus kit (Millipore) and exposed by autoradiography (Eastman Kodak, Rochester, NY, USA).