Precision plus standards

Cell suspensions (2.5x10⁷ cells/ml) were solubilised in modified RIPA buffer (1% Triton X-100, 0.25% sodium deoxycholate, 0.15M NaCl, 50mM Tris-HCl, 5mM EDTA containing Protease Inhibitor (Roche, Basel, Switzerland)). Protein content was determined by bicinchoninic acid assay (ThermoFisher). Lysates (5μg) were fractionated on a 4–12% Bis-Tris gel (Bolt, ThermoFisher) under reducing conditions. Proteins were transferred to nitrocellulose (Novex Miniblot; ThermoFisher) using an iBlot. Membranes were stained with Ponceau before overnight incubation in 5%BSA in TBST. Membranes were incubated with 1μg/ml rabbit anti-human CD302 polyclonal antibody (LS-C119435; LS Bio) followed by 1:1000 HRP conjugated goat anti-rabbit IgG Fc antibody (A6154 Sigma-Aldrich). Protein bands were detected by chemiluminescence using Clarity Western ECL substrate (BioRad) and visualised on a BioRad GelDoc. Molecular weights (MW) of proteins were determined by comparison with Precision Plus standards (BioRad).

Serum was diluted in PBS and heated for 15 min at 90 °C before being electrophoresed on 15% SDS/acrylamide gels, followed by a transfer onto a 0.2 µm nitrocellulose membrane (BioRad, Hertfordshire, UK) for 1 h at 4 °C. Membranes were blocked for 1 h in blocking buffer (5% milk in TBS, 0.1% Tween-20) before incubating with anti-Haptoglobin (Sigma-Aldrich, Poole, UK HYB 170-06-02) diluted in block buffer overnight at 4 °C. Membranes were washed for 2 × 5 min in wash buffer (88mM Tris pH7.8, 0.1% Tween-20), prior to incubating with species-specific horse radish peroxidise conjugated goat anti-mouse secondary antibody (New England Biolabs, Hertfordshire, UK) for 1 h at room temperature. Membranes were washed (3 × 5 min) in wash buffer, and immunoreactive proteins were visualised using enhanced chemiluminesence in the BioRad Universal Hood II with Quantity One Software. Haptoglobin isolated from human plasma (Sigma-Aldrich, Poole, UK) was used as a reference, and protein molecular weights were determined using Precision Plus Standards (BioRad, Hertfordshire, UK).