As previously described 54, immunoprecipitations were performed with an anti-CD16 mAb (clone 3G8, mice IgG1, BD biosciences) or an anti-EGFR mAb (mice IgG1, Santa Cruz, Heidelberg, Germany) and sera of
non-immunized mice (Dako) used as the negative control. Immunoblotting was performed using Nupage selleck kinase inhibitor system (Invitrogen) and L1 proteins from VLPs were detected using CAMVIR antibody (Abcam, Cambridge, UK) and Clean Blot IP detection reagent (Thermo Fisher). The assay to detect activated GTPase proteins was carried out as previously described 55. Briefly, cells were lysed by addition of 200 μL of ice-cold lysing buffer. Lysates were centrifuged for 5 min at 16 000×g. Supernatants were immediately frozen in liquid nitrogen and stored at −80°C until use. For pull-down assays, supernatants were incubated for 30 min with 30 μg of GST-PBD protein containing the Cdc42 and Rac1 binding regions of PAK-1B, affinity linked to glutathione-sepharose beads. The beads were washed in ice-cold washing buffer and boiled in SDS-PAGE lysis buffer. The amount of Rac1 and Cdc42 in the samples was https://www.selleckchem.com/products/Romidepsin-FK228.html determined by immunoblotting with antibodies specific to Rac1 (23A8, Upstate Biotechnology,
Waltham, USA) and Cdc42 (BD Biosciences). Prism 4.0 (GraphPad Software) was used for data handling, analysis and graphic representation. Statistical analysis was performed using Student’s t-test or the Mann–Whitney test. The authors thank Dr. S. Ormenese from the GIGA-Imaging and Flow Cytometry platform for her support with flow cytometry and confocal microscopy and Prof. N. Antoine for the preparation of electron microscopy grids. They are also grateful to Dr. P. Coursaget for the provision of baculovirus expressing HPV16 and HPV31 L1, Dr. L. Bousarghin for providing electron microscopy grids with DCs containing HPV-VLPs, Prof. N. Christensen for providing
V5 antibodies, Immune system M. Lebrun for her assistance with confocal microscopy, Dr D. Begon for her advice on co-immunoprecipitation and Prof. G. Thibault for helpful discussion. They thank GlaxoSmithKline Biologicals for providing polyclonal antibodies used to assess the quality of L1-VLPs by ELISA. This study was supported by the Belgian National Fund for Scientific Research (FNRS), C. D., A. C. and N. J. are supported by the FNRS. V. R., B. B. and I. L. are supported by a Télévie grant from the FNRS. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors.