Ab6514

Cultured cells were infected with HSVQ, HSV-P10, or saline at the indicated MOI for the indicated time. Cell lysates were harvested in RIPA cell lysis buffer with protease and phosphatase inhibitors added prior to use. Lysed cells were sonicated and cell debris removed via centrifugation. Protein concentration was measured by BCA and normalized prior to loading in precast gels. PVDF membranes were exposed to antibodies following manufacturer’s protocol. Primary (from CST: PTEN (9559), Akt (4691), pAkt-S473 (9271), from abcam: HSV1 ICP4 (ab6514), GAPDH (ab9484)) and secondary antibodies (from abcam: anti-mouse HRP (ab6789), anti-rabbit HRP (ab6721) can be found in Supplementary Data 1. All phospho-antibody incubations were performed overnight at 4 °C, and all non-phospho-antibody incubations were performed at RT for 1–2 h. Uncropped blots are depicted in Supplementary Figure 9.

The HSV-1(MOI = 0.01) infected HEp-2 cells were in the presence or absence of R. tanguticum nanoparticles (350 µg/ml). At 6, 12, 18, and 24 h.p.i., the cells were fixed with pre-chilled 4% paraformaldehyde for 30 min and were permeabilized in PBS containing 0.25% Triton X-100 for 10 min at room temperature and then rinsed thrice in PBS, following by blocked in 1% BSA for 30 min. Cells were further washed with PBS, then incubated for 2 h at 37°C with anti-ICP4 antibody (Abcam, ab6514, 1:1000) and anti-ICP8 (Abcam, ab20194, 1:500) antibody, respectively. The cells were then rinsed three times for 5 min in PBS, followed by incubation with the 488-conjugated goat anti-mouse IgG (H+L) secondary antibody (diluted 1:200) for 60 min at 37°C and counterstained with DAPI. Images were observed using a fluorescence microscope (TE2000; Nikon, Tokyo, Japan) (Roberts and Baines, 2011 (link)).

NPCs were grown to 85–90% confluence in matrigel-coated 96-well plates and infected with acyclovir-resistant HSV-1 strain tk- or PAAv at MOI 0.1. At 24 hours post-infection cells were fixed in 4% PFA, permeabilized with 0.2% Triton X-100, stained with antibody to ICP4 (Abcam ab6514) followed by alexa-488 anti-mouse secondary, and counterstained with Hoechst 33342. Cells were imaged with Nikon AS1 microscope and counted using Nikon NIS-elements software. IC50 values for ACV and R430 against mutant virus strains and KOS control strain were calculated using 4-parameter log logistic curve implemented using the DRC R package.

HEp-2 cells were infected with HSV-1 at an MOI of 0.01 and were incubated with or without R. tanguticum nanoparticles (350 µg/ml) at intervals of 6, 12, 18, and 24 h post-infection. Cells were harvested in RIPA Lysis Buffer (Biosharp, Hefei, China) and the soluble fraction was then clarified by centrifugation at 12,000 × g for 5 min at 4°C. Equal amounts of protein (40 µg/sample) were then isolated by 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a pre-equilibrated PVDF membrane (Thermo Scientific). Membranes were blocked with 5% BSA for 2 h, rinsed and followed by incubation with anti-ICP4 antibody (Abcam, ab6514, 1:1000), anti-ICP8 antibody (Abcam, ab20194, 1:500) and anti-GAPDH antibody (Tianjin Sungene Biotech KM9002T, Beijing, China, 1:5,000) in 5% BSA at 4°C overnight, respectively. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature and visualized by using an ECL Western Blot Detection Kit (Millipore Corp., Bedford, MA).

Antibodies against ICP8 (Rumbaugh Goodwin Institute for Cancer Research, Inc.), ICP0 (Cat No. ab6513; Abcam), ICP4 (Cat No. ab6514; Abcam), ICP27, VP16, and US11 have been described elsewhere (Ackermann et al., 1984 (link); McKnight et al., 1987 (link); Roller and Roizman, 1992 (link)). Antibodies against ICP22, VP22, and VP16 were kind gifts of Bernard Roizman (The University of Chicago, United States). Additional antibodies used in this study were anti-green fluorescent protein (anti-GFP) monoclonal antibody (Cat No. KM8009; Sungene Biotech) and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Cat No.KM9002; Sungene Biotech).