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Fig. 2
Identification of candidate 4C4 antigens by proteomic analysis. (A) Antigen identification strategy, from sample preparation to LC-MS analysis. (B) Immunoprecipitation (IP) of the target protein detected by western blot using the 4C4 antibody. Full blot showing: IP using the control isotype antibody IgGk1 (IP IgG), IP using the 4C4 antibody (IP 4C4), brain lysate as a positive control (lysate). The two bands detected in the IP samples correspond to the heavy and light chains (~50 and 25 kDa) of the primary antibody that are being recognized by the secondary antibody. M, protein marker; kDa, kilodaltons. (C) Volcano plot of averaged enrichment of protein in 4C4 (IP 4C4) vs. IgGk control (IP IgG CTL) immunoprecipitations. Red dots: enriched proteins (Fold change ≥ 2) P-value < 0.05. (D) Reproducibility boxplot of the total number of spectral counts quantified in each IP linked by pair (n = 5 independent experiments) for the three most significantly enriched proteins are shown. ***P < 0.001, **P < 0.01, *P < 0.05; paired t-test. LC-MS/MS, liquid chromatography-tandem mass spectrometry
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