Described. two.9. Confocal Microscopy To evaluate the internalization of Nef protein by Confocal Laser Scanner Microscopy evaluation, main human pDCs and GEN2.two cells have been seeded at 105 cells/200 and 0.2 106 cells/150 , respectively, in full ten FBS medium in 96-well plates and treated with 300 ng/mL of myrNefSF2 w.t-Alexa488 or myrNefSF2 4EA-Alexa488, which were labelled applying AlexaFluor488 Microscale Protein Labelling Kit (IL-15R alpha Proteins web Molecular Probes/Invitrogen, Monza, Italy) following the manufacturer’s recommendations. Cells have been harvested at indicated instances, washed as soon as in 1PBS, placed around the microscope slide and left to air dry. Subsequently, they were fixed with 4 PFA for 15 min on ice and after that washed three instances with PBS. Finally, coverslips have been mounted employing Vectashield antifade mounting medium (Vectashield H-1000; Vector Laboratories Inc., Burlingame, CA, USA) diluted at 80 in PBS to prepare samples for confocal microscopy observation. Plasma membrane counterstaining was performed by treating major pDCs for five min with PKH26-GL, using the PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma-Aldrich, Milan, Italy) following the manufacturer’s suggestions. Nuclei of GEN2.2 cells have been stained with 3 /mL DAPI (4 , six -diamidino-2-phenylindole) (Sigma-Aldrich, Milan, Italy) that was straight added to the mounting medium. In an effort to assess IRF-7 enhance, key pDCs have been seeded at 105 cells/200 in total 10 FBS medium in 96-well plates and treated with myrNefSF2 w.t (300 ng/mL) or CpG A (3 /mL). Major pDCs had been fixed with 4 PFA for 15 min on ice, then washed three times with PBS and permeabilized with 0.1 Triton X-100 in PBS for ten min on ice. Afterwards, the specimens were incubated for 30 min in the dark at RT with 1 BSA in PBS containing far-red fluorescent dye RedDotTM2 to stain nuclei (Biotium, Inc. Hayward, CA, USA), washed and after that incubated within the dark for 1 h at RT with all the following antibodies: rabbit anti-IRF-7 antibody (Santa Cruz Biotechnology, Dalls, TX, USA, cat. #sc-9083), diluted 1:50 in 0.1 BSA in PBS, and AlexaFluor546-conjugated anti-rabbit (Life Technologies, Monza, Italy, cat. #A11010) as a secondary antibody, diluted 1:200 in 0.1 BSA in PBS. Lastly, the specimens have been washed four instances in PBS and ready for confocal microscopy observation, as previously described. For pulse-chase studies, 3 105 GEN2.two cells were seeded in 48-well plates and metabolically labelled with Bodipy C16 in accordance with the concentrations and Integrin alpha-5 Proteins Biological Activity interval of instances reported. Cells had been then washed twice with 1PBS, placed on a microscope slideViruses 2022, 14,8 ofand fixed as reported above. Lastly, samples were mounted with Vectashield antifade mounting medium containing DAPI for nucleus staining. All samples were stored protected in the light at 0 C till the observation. Photos had been acquired with Leica TCS SP5 confocal microscope and processed with LAS AF application (version 1.6.three, Leica Microsystems CMS GmbH). Objective 63.0X. Lasers activated: Argon laser at 488 nm to visualize myrNefSF2 -Alexa488 (green) and UV laser at 405 nm to observe nuclei stained with DAPI. Images have been acquired activating single laser in sequential mode to prevent fluorescence overlay. Many fields had been analysed for every single condition and representative benefits are shown. two.ten. RNA Extraction and Quantitative RT-PCR Evaluation For RNA extraction, cells have been seeded at 106 cells/mL and treated for 6 h with 300 ng/mL of my.