As expected, no significant variations were observed in the total content of PKR throughout the duration of the infection

May 15, 2017

nd whether the different TM domains have an effect on scFvFc function, 293T cells were transiently transfected and harvested as described above followed by incubation with biotinylated Tat Recognition Motif peptide at 10 10381773 mM final concentration/sample on ice for 30 minutes and washed 36 Generation of scFvFc-TM/VSV-G pseudotyped lentiviruses and 293T cells stably expressing scFvFc-TM through lentiviral transduction A self-inactivating pHAGE lentivector was modified to accommodate subcloning of Sfi-I/Pac-I scFvFcTM DNA fragments. pHAGE lentivector has a CMV promoter that drives the expression of the transgene. It also expresses the Zoanthus Green Fluorescent protein gene via an IRES Antibody Display and Discovery sequence, which can be used to monitor and normalize transduction efficiencies. For the production of VSV-G pseudotyped lentiviral particles, total of 56106 293T cells were seeded on 100 mm diameter plates, and co-transfected the next day with 10 mg of the pHAGE lentivector and packaging plasmids, using the CaPhosphate method. At 48 and 72 hours post transfection, supernatant was collected and cleared by centrifugation and sterile filtered through a 0.45 mm filter. Viral supernatant was concentrated by ultracentrifugation through a 20% sucrose cushion, aliquoted and stored at 280uC. The titer of the pseudotyped virus particles was evaluated either by RT assay or by transduction of HeLa cells with increasing dilutions of the lentivirus stock and measurement of ZsGreen marked cells or APC-anti-human Fc staining by FACS analysis. Importantly, incorporation of the scFvFc-CD28/CD28gp41 fusion protein did not lower the titer of viral particles. 293T cells were transduced, in the presence of 8 mg/ml polybrene, with above generated recombinant lentiviruses at different MOI as indicated for 4 hours. Functional scFvFc expression on the transduced cell surface was analyzed by FACS, following a similar protocol as described above, first at 48 hours post-trandsuction then again after longer periods of propagation to confirm stable scFvFc expression. Biotinylated peptide or protein antigens purchase beta-Mangostin 9776380″ target=_blank”>9776380 and their concentration used for specific antibody binding analysis are described in the figure legends. with 2% BSA and 0.05% saponin for 30 minutes and incubated with either a mouse anti-p24 antibody or with an anti-human Fc-rhodamine antibody for 1 hour. Cells were then washed and incubated with Cy2-labeled antimouse IgG antibody for an additional hour. Controls include virus-bound cells incubated with the secondary antibody alone. Finally, samples were mounted for fluorescence microscopy by using ProLong antifade kit. Images were acquired by using BIO-Rad Radiance 2000 laser scanning confocal microscope using a Nikon 606. ScFvFc quantification on the surface of transfected or transduced cells To quantify the number of scFv-Fc molecules on the surface of transiently transfected or lentivirus transduced cells, the ��Quantum Simply cellular anti-mouse IgG kit��was used. Briefly, cells expressing on their surface the scFvFc with a C9 tag were incubated with 10 mg of an APC conjugated mouse antiC9 monoclonal antibody 1D4 for 1 hour on ice in PBS supplemented with 2% BSA. Calibration beads with known binding capacity of mouse IgG molecules on their surface were treated with the same conditions as the cells. Upon washing, both cells and calibration beads were analyzed for APC staining intensity by FACS. Number of scFv-Fc molecules on cell surface was determined b