Wet method

Western blot was conducted as described by Abdi et al. [33 (link)]. Briefly, the SUSP were separated by electrophoresis and transferred to nitrocellulose membrane (Bio-Rad) at a constant 100 V with 400 mA for 60 min by a wet method (Bio-Rad). The membrane was blocked overnight, washed, and incubated with hyperimmune serum from a cow previously vaccinated with SUSP and protected from mastitis upon challenge in another previous pilot study. The membrane was washed and incubated with horse radish peroxidase (HRP)-conjugated sheep anti-bovine IgG (H+L) secondary antibody (Bethyl Lab. Montgomery, TX, USA). The precision protein Strep Tactin-HRP conjugate (Bio-Rad) was used as a secondary antibody for molecular weight markers. Finally, the membrane was washed and 25 mL of peroxidase substrate (TMB membrane HRP substrate (SeraCare Life Sciences Inc, Milford, MA, USA) was added, and the reaction was fully developed at room temperature. Protein band images were taken using a ChemiDoc™ Touch Imaging System (Bio-Rad) and analyzed using Image Lab Software (Bio-Rad). Images on the gel and the membrane were compared to identify immune-reactive protein bands.

Cells were lysed directly in 1x Laemmli buffer and proteins were separated through denaturating SDS-PAGE electrophoresis using Mini-Protean TGX precast gels (Biorad) and transferred on Nitrocellulose membrane using the wet method (Biorad). Membranes were blocked with 5% skim milk in TBS-1% Tween (TBST) 1h at room temperature and probed overnight at 4°C with primary antibodies in TBST 2% milk. The following antibodies were used: rabbit anti-MRTF-A (Abcam, ab49311, 1/500), rabbit anti-pan actin (Cytoskeleton, AAN01-A, 1/750) and mouse anti-hsc70 (Santa Cruz, SC7298, 1/1 000). Following washing in TBST, membranes were hybridized with goat anti-mouse and goat anti-rabbit secondary antibodies coupled to HRP (ThermoFisher, 62–6520 and A27036, 1/10 000). Proteins were revealed using SuperSignal West Femto substrate (ThermoFisher). Proteins were quantified by using FusionCapt Advance software (Vilber Lourmat).