Milk powder

Proteins were resolved by electrophoresis on 12% SDS-PAGE gels using a mini-Protean Tetra cell system (Bio-Rad). Electrophoresed proteins were transferred to a nitrocellulose membrane (Bio-Rad) using a mini-Transblot electrophoretic transfer cell (Bio-Rad). Membranes were blocked in 1× Tris-buffered saline (TBS; Severn Biotech) containing 0.001% (vol/vol) Tween 20 (Acros Organics) and 5% (wt/vol) fat-free milk powder (Sainsbury’s). c-Fos, p-DUSP1/MKP1, and α-actin primary antibodies were diluted (1:3,000, 1:1,000, and 1:10,000, respectively) in TBS-Tween and 5% milk, and membranes were incubated overnight at 4°C with gentle shaking (c-Fos and p-DUSP1/MKP1) or for 1 h at room temperature with gentle shaking (α-actin). Following incubation, membranes were washed with 1× TBS containing 0.001% (vol/vol) Tween 20, diluted (1:10,000) horseradish peroxidase (HRP)-conjugated secondary antibody was added, and membranes were incubated for 1 h at room temperature. Membranes were washed as described above and exposed to the Immobilon Western chemiluminescent HRP substrate (Millipore) prior to visualization by exposure to film (GE Healthcare). α-Actin was used as a loading control.

Proteins were resolved by electrophoresis on 12% SDS-PAGE gels using a mini-Protean Tetra cell system (Bio-Rad). Electrophoresed proteins were transferred to a nitrocellulose membrane (Bio-Rad) using a mini-Transblot electrophoretic transfer cell (Bio-Rad). Membranes were blocked in 1× Tris-buffered saline (TBS; Severn Biotech) containing 0.001% (vol/vol) Tween 20 (Acros Organics) and 5% (wt/vol) fat-free milk powder (Sainsbury’s). Primary antibodies diluted (1:1,000) in TBS-Tween and 5% milk (c-Fos) or TBS-Tween and 5% bovine serum albumin (p-DUSP1/MKP1) were added, and membranes were incubated overnight at 4°C with gentle shaking. Following incubation, membranes were washed with 1× TBS containing 0.001% (vol/vol) Tween 20, diluted (1:10,000) horseradish peroxidase (HRP)-conjugated secondary antibody was added, and membranes were incubated for 1 h at room temperature. Membranes were washed as described above and exposed to Immobilon Western chemiluminescent HRP substrate (Millipore) prior to visualization by exposure to film (GE Healthcare). Alpha-actin was used as a loading control.

ELISA plates (Greiner Bio-One B.V, Alphen aan den Rijn, the Netherlands) were coated with 250 ng of the antigens in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6–9.8) and incubated for three days at 4°C. The plates were blocked for 45 min at 37°C in coating buffer with 2.5% milk powder (Oxoid, Hampshire, UK). After washing, serial twofold dilutions of the sera were made in PBS containing 0.05% Tween-20 (PBST) and incubated for 2 h at 37°C. After washing with PBST, the plates were incubated with GaM/IgG-HRP (SouthernBiotech) (1:5000 in PBST) for 90 min at 37°C. Finally, the peroxidase reaction was visualized using o-phenylene-diamine (Sigma-Aldrich) for 30 min at room temperature. The reaction was stopped by adding 2 M H₂SO₄. The plates were measured at 492 nm in a plate reader (Biotek Powerwave XS2, Beun de Ronde, Abcoude, the Netherlands).