Recently, Silvie et al. have described the MT81w mAb, which specifically recognizes mouse

CD81 PARP inhibitor associated with other tetraspanins. This is evidenced by the lack of recognition of CD81 after cell lysis with detergents that do not preserve tetraspanin-tetraspanin interactions, and by the complete removal of the CD81 pool recognized by MT81w following immunodepletion of tetraspanin complexes [23]. CD81 is required for invasion of hepatocytes by sporozoites of human malaria Plasmodium falciparum and rodent malaria Plasmodium yoelii parasites [26]. Using MT81w antibody, Silvie et al. have shown that the subset of CD81 associated with TEMs contributes to Plasmodium sporozoite infection [23]. Such an antibody preferentially recognizing human CD81 associated with TEMs is not available. However, since Huh-7w7/mCD81 cells are susceptible to HCVcc and HCVpp-2a infection, we next took advantage of this model and the MT81w mAb to study the role of TEM-associated CD81 in the early steps of HCV life cycle. Using the MT81w anti-mCD81 mAb, we first characterized the subpopulation of mCD81 that is associated with TEMs on the cell surface of Huh-7w7/mCD81 cells (Figure 3A). As shown by flow cytometry

analysis, the intensity of MT81w labeling only reached 32 ± 14%, depending on the experiment, of the intensity with MT81 in Huh-7w7/mCD81 cells, indicating that only a fraction of CD81 molecules is engaged in tetraspanin microdomains on these cells, as described for Hepa1–6 cells [23]. However, we cannot exclude that the lower affinity of MT81w may lead to an underestimate Q-VD-Oph manufacturer of the ratio of CD81 engaged in TEMs. The specificity of MT81w to recognize TEM-associated CD81 in Huh-7w7/mCD81 cells was confirmed by immunoprecipitation experiments from biotinylated

cell lysates made under different detergent conditions. Tetraspanin microdomains Dehydratase are typically disrupted by Triton X-100, but are retained in less hydrophobic detergents such as Brij 97 [30]. As shown in Figure 3B, 5A6 and MT81 mAbs precipitated hCD81 and mCD81, respectively, under both detergent condition. In contrast, MT81w was able to Trichostatin A cell line precipitate mCD81 only from Brij97 lysates preserving tetraspanin-tetraspanin interactions, but not from Triton X-100 lysates. These results show that expression of mCD81 in Huh-7w7 cells allowed to reconstitute tetraspanin webs that are specifically recognized by the well characterized MT81w mAb [23]. Figure 3 Recognition of TEM-associated CD81 in Huh-7w7/mCD81 cells. A, Flow cytometry analysis of Huh-7w7/mCD81 cells stained with the mAbs MT81 and MT81w. Cells stained only with PE-conjugated secondary antibody were used as control (dotted line). B, Cell lines were surface biotinylated and lysed in the presence of Brij97 or Triton X-100 before immunoprecipitation with MT81, MT81w and 5A6 mAbs. Immunoprecipitates were revealed by western blotting using peroxidase-conjugated streptavidin.