Agarose

Immunoprecipitation was carried out to assess protein–protein interaction^{50 (link)}. Briefly, HEK293T or THP-1 cells were infected with HSV-1 or VSV (MOI = 0.5) for 16 h. Cells were harvested and lysed with NP40 buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) supplemented with a protease inhibitor cocktail (Roche). Centrifuged cell lysates were precleared with Sepharose 4B beads and incubated with 10 μl of FLAG-Agarose (Sigma-Aldrich) or 0.5 μg of the indicated antibody plus 10 μl of protein G-Sepharose (GE Healthcare) at 4 °C for 4 h. Agarose beads were washed three times with lysis buffer, and precipitated proteins were released by boiling with 1× sodium dodecyl sulfate (SDS) sample buffer at 95 °C for 5 min. Precipitated proteins were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) and analyzed by immunoblotting.

H1299 cell lysates were prepared using NP-40 lysis buffer. Cap-binding reactions were performed at 4 °C with 1 mg protein and 15 ml m⁷GTP agarose or agarose (GE Healthcare) in NT2 buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MgCl2, and 0.05% NP40] for one hour. Binding reactions were performed in the presence or absence or 0.1 mM cap analogues GpppG. Sepharose beads were washed three times with 1 ml NT2 buffer and then resuspended in sample buffer for SDS–PAGE. Antibodies to eIF4A (Abcam), eIF4G (Abcam), 4E-BP1(Cell signaling) and eIF4E (Cell signaling) were used for western blotting.

The protocol used was similar to that previously described by our group^{1 (link)}, but we introduced the modifications described below.
We incubated 40 µL of CSF and serum samples with 5 µL of 0.5 M dithiothreitol and 5 µL of 1 M Tris/HCl pH 9.7, for 45 min.
For protein separation, we used an agarose gel consisting of 0.3 g of agarose (GE Healthcare); 3.6 g of sorbitol (Sigma-Aldrich); 1.25 mL of each Pharmalyte, pH 4–6.5 and pH 3–10 (GE Healthcare); 2.5 mL of glycerol, and 22.5 mL of water.
To detect IgA, we used a biotinylated anti-human IgA antibody (Jakson Immunoresearch) diluted (1:20.000) in the blocking solution. After incubating the membrane overnight at 4 °C, the labeling was developed using streptavidin-HRP (Jakson Immunoresearch) diluted (1:1.000) in blocking solution.
To study the sensitivity of this new assay (IEF and immunodetection), we analyzed paired CSF and serum samples from 3 MS patients showing OGIgAB. We applied 5 µL of twofold serial diluted samples (from 2 to 0 ng of IgA) on the gel in triplicate.
Representative full blots including the patterns of OGIgAB represented in pictures 1 and 2 are supplied in Supplementary Data 1.
To assay the limit of blank and the limit of detection, diluted CSF and serum samples with an extremely low quantity of IgA (0 ng and 0.25 ng respectively) were analyzed.