Selection of intrabodies that inhibit mast cell degranulation.

<p>a) Schematic view of the selection method. The scFv/intrabody library previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104998#pone.0104998-Philibert1" target="_blank">[13]</a> was cloned in plasmid and retroviral vectors and used to transfect the RBL-2H3 cell line in order to induce a phenotypic diversity in a collection of cells. Clones displaying the desired phenotype, measured by inhibition of degranulation, were selected and the couple constituted by the inhibitory intrabody and its target antigen was identified and characterized. b) Annexin-V staining of cell populations from the library selection rounds is illustrated as the ratio of the geometric mean (MFI) of the FcεRI-stimulated (S) to the unstimulated (NS) cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104998#pone.0104998.s001" target="_blank">Fig. S1</a>).</p

Anti-C12orf4 intrabody inhibits mast cell degranulation.

<p>Analysis of stable clone 5H4: a) measurement of Annexin-V staining; b) β-hexosaminidase release; c) calcium flux and d) TNFα secretion. T-: Irrelevant intrabody. S: IgE/DNP stimulated. NS: unstimulated. Boxplot whiskers extend to the most extreme data point that is no more than 1.5 times the interquartile range. e) Measure of β-hexosaminidase release by retroviral infected populations. Clone R_8 is identical to the intrabody expressed by the plasmid clone 5H4. Sequences of the clones are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104998#pone.0104998.s006" target="_blank">Fig. S6</a>. Boxplot whiskers extend to the most extreme data point. f) Specific binding of 5H4-VH to C12orf4. Top panel: pull-down assay using 5H4-VH as capture agent and a commercial anti-C12orf4 polyclonal serum to reveal the protein. Irr: Irrelevant VH fragment, differing from 5H4 VH only by its CDR3 sequence. Low panel: subcellular localization of C12orf4 analyzed by confocal laser microscopy after double staining. Top left: Hoechst; top right: 5H4-VH-Fc fusion; bottom left: anti-C12orf4 commercial antibody; bottom right: merge. *: p<0.05; **: p<0.01; ***: p<0.001 (Student t-test).</p