Foxo3a 2497

For Western blotting and immunofluorescence imaging: HSV-1 viral proteins were detected by ICP0 (ab6513) 1:1000, gB (ab6506) 1:1000 both purchased from Abcam (Cambridge, United Kingdom. Following antibodies were used for western blot or immunofluorescence were FoxO3a (2497S) 1:1000, AKT (9272S) 1:1000, p-FoxO3a^S253 (9466S) 1:500, Rictor (2114S) 1:1000, p-FoxO1^T24/p-FoxO3A^T32 (9464S) 1:500, p-AKT^S473 (9271S) 1:500, FoxO1 (2880) 1:1000, IRF7 (4920) 1:1000 all purchased from cell signaling technology (Danvers, MA), GAPDH (10494-1-AP) 1:2000 was purchased from Proteintech Group, Inc., (Rosemont, IL).
For flow cytometry: Flow antibodies were purchased from Biolegend were CD3 (100236), CD69 (104507), CD11b (101206), CD11c (117310), CD49b (108907), CD317 (127104) and those purchased from Tondo biosciences, San Diego were CD4 (50-0042-U100) and CD8a (20-18886-U100).

Cells were lysed in NP-40 lysis buffer (50 mM HEPES [pH 7.5], 150 mM KCl, 2 mM EDTA, 1 mM NaF, 0.5% [vol/vol] NP-40, 0.5 mM dithiothreitol). Protein concentrations were determined using the bicinchoninic acid protein assay kit (Thermo Scientific), and 20 μg of total protein lysate was resolved on 10% Tris-glycine SDS-PAGE and then transferred onto Immobilon polyvinylidene difluoride membranes. Blots were probed with primary antibodies to Zeb2 (sc-271984; Santa Cruz), Foxo3a (2497S; Cell Signaling), phospho-Foxo3a Ser294 (5538S; Cell Signaling), RanBP9 (17755-1-AP; Proteintech), EBV Ea-D (BMRF1, sc-0261; Santa Cruz), EBV Zebra (BZLF1, sc-53904; Santa Cruz), BHRF1 (biorbyt orb518144), GAPDH (sc-47724; Santa Cruz), or beta-actin (sc-47778; Santa Cruz), followed by horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG). Blots were developed with enhanced chemiluminescent substrate (Pierce). Band intensities were quantified using ImageJ, normalized to loading controls, and reported relative to control cells.

Cells were washed in PBS and then lysed. The protein concentration of each cell lysate was determined and equated. Prepared samples were boiled in sample buffer at 100 °C for 5 minutes. Samples were loaded in SDS-PAGE gel and the gel ran at 140–160 V. After electrophoresis proteins were transferred to the PVDF membrane. Membranes were blocked for 1.5 h at room temperature using blocking buffer (5% nonfat dry milk) and incubated with primary antibodies for 1 h at room temperature or overnight at +4 °C. Then membranes were incubated with HRP-conjugated secondary antibodies diluted in blocking buffer for 1h at room temperature. As primary antibodies, we used antibodies to Foxo1 #2880, Foxo3a #2497, phospho-Foxo3a #9466, Akt #9272, phospho-Akt #4060 (Cell Signalling).