Seeblue 2

20 μg of ERGvir and ERGatt protein samples were loaded on pre-cast NuPage 4–12% Novex Tris-Glycine Gels, as described elsewhere [29 (link)]. Briefly, samples were solubilized in Laemmli buffer, under reducing conditions, and subjected to electrophoresis for 35 minutes in NuPage MES Buffer (all from Invitrogen, UK). Each gel included pre-stained molecular mass markers (SeeBlue 2, Invitrogen, UK). Proteins were stained with Coomassie/Colloidal Brilliant Blue R-250 and 14 gel bands were cut across the three ERGvir and three ERGatt biological replicates lanes (S3 Fig).

Bromelain-digested PR8 haemagglutinin (HA) was prepared as previously described [66] (link). P. chabaudi-iRBC were solubilized in Triton X-100 and SDS buffer in the presence of protease inhibitors [67] (link). Proteins were resolved by electrophoresis through NuPAGE 4–12% acrylamide gels in MES buffer (Invitrogen) under reducing conditions. Markers were the broad-range pre-stained protein standard Seeblue2 (Invitrogen). Proteins were transferred onto Hybond C extra nitrocellulose membrane (Amersham Pharmacia), as described previously [68] (link). Specific proteins were detected using (i) unpurified sera from influenza immune mice or (ii) unpurified sera from P. chabaudi infected mice, followed by Alexa 680-conjugated goat anti-mouse IgG (1∶15,000) (Licor Biosciences) and revealed by scanning the membranes with the Odyssey scanner (Licor Biosciences) using 680EX nm/700EM nm filter settings. Plasma from uninfected mice was used as a negative control.

Gels were blotted to nitrocellulose membranes (Protran83; Schleicher & Schuell) for 1 h constantly at 300 mA, blocked for 1 h at room temperature and incubated for 1 h with the anti-His₅ horseradish peroxidase-conjugated antibody following the manufacturer's instructions (Penta-His HRP Conjugate Kit, Qiagen). Membranes were washed twice in TBS-Tween-Triton buffer and once in TBS buffer for 10 min each. Detection was followed by incubating with enhanced chemiluminescence Prime Western Blotting Detection Reagent (GE-Healthcare). As a molecular weight standard, Seeblue 2 (Invitrogen) was used. For the purified domains, western blotting intensities were analysed using the integrated density function of ImageJ (https://imagej.nih.gov/ij/index.html).