doi: 10.1128/JVI.78.15.7969-7983.2004. a novel cellular factor, gp96, that is critical for both HHV-6A and -6B entry into host cells. As gp96 can function as an adjuvant in vaccine development for both infectious agents and cancers, it can be a potential therapeutic CHR-6494 target for infection by these two viruses. cells (BL21 strain) and used to immunize mice. Mouse IgG isotype control (A7028; Beyotime) and mouse monoclonal antibodies against -actin (60008-1; Proteintech), CD46 (J4.48; Abnova Corporation), CD134 (ACT35; CST), and Flag (F1804; Sigma) were purchased. Coimmunoprecipitation. Antibodies were bound to protein G-Sepharose (GE Healthcare) by incubation at 4C for CHR-6494 8 h and then cross-linked with protein G-Sepharose with dimethyl pimelimidate (DMP; Thermo Scientific) according to the manufacturers instructions. The cells were lysed in TNE buffer (10?mM Tris HCl [pH 7.8], 0.15 M NaCl, 1?mM EDTA, and 1% Nonidet P-40 [Nacalai Tesque] containing protease inhibitor cocktail [Sigma-Aldrich]) for 30?min at 4C. After centrifugation at 13,000??for 1 h at 4C, the supernatant was incubated with the appropriate protein G-Sepharose-bound antibody at 4C for 8 h. The bound proteins were eluted with 0.1 M glycine (pH 2.8) at 4C, collected, and neutralized with 1 M Tris-HCl (pH 9.0) to pH 7.0 to 7.4. The samples were suspended in 5 SDS sample buffer, boiled for 5?min at 100C, and then analyzed by Western blotting with the indicated antibodies. Mass spectrometry. The above immunoprecipitates were electrophoretically separated in a denaturing gel and visualized with silver staining. The remaining immunoprecipitates were digested with trypsin and analyzed using mass spectrometry analysis on an AB Sciex TripleTOF 5600+ system (Novogene, China) (54). Western blot analysis. Samples were separated by SDS-PAGE, electrotransferred onto polyvinylidene difluoride (PVDF) membranes, and reacted with primary antibodies. The reactive bands were detected using horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) and visualized using enhanced chemiluminescence (ECL) reagents. Establishment of cell lines stably expressing shRNA against human gp96. A gp96 shRNA-expressing lentivirus and its control were generated by transfecting 293T cells with packaging plasmids (pCAG-HIV-gag and pCMV-VSV-G-RSV-Rev) and the pLKO.1gp96 plasmid or a pLKO.1 control plasmid. The culture media containing the viruses were harvested at 3 Mouse monoclonal to HER-2 days after transfection and centrifuged at 12,000??for 1 h. Cells were transduced with the lentiviruses for 1 day and selected with puromycin (0.2 to 1 1?g/ml) in maintenance medium. Transferrin uptake assay. Control and gp96 knockdown cells were treated with 50?g/ml Alexa 488-labeled transferrin (“type”:”entrez-protein”,”attrs”:”text”:”T13342″,”term_id”:”7515367″,”term_text”:”pirT13342; Thermo Scientific) for 1 h at 4C and then incubated at 37C for 15?min. After being washed with citric acid buffer (0.1 M, pH 3.0), the cells were analyzed with FACS analysis. Quantitative real-time PCR analysis for viral genome quantification. Factor gp96 knockdown or corresponding control cells CHR-6494 (5??105 cells for each sample) were infected with HHV-6 for 2 h at 37C and then washed with 0.25% trypsin-EDTA. Total DNA was extracted from the infected cells with a nucleic acid extraction kit (DP304; Tiangen). Viral DNA was quantified using U22 primers (5-CGCTCGGAAAGGAAACATTA-3 and 5-AAGTGGAACTGCTTGGTGGC-3) on a 7500 fast PCR system (Applied Biosystems) using TB Green Premix Ex Taq (TaKaRa). The data were normalized to the expression of actin (forward primer: 5-TGGCACCCAGCACAATGAA-3, reverse primer: 5-CTAAGTCATAGTCCGCCTAGAAGCA-3). Pulldown assay. The secreted form of the gH/gL/gQ1/gQ2 complex (The ectodomain CHR-6494 of gH was tagged with Fc and 6 His.) and the Fc protein were purified from the culture medium of their expression cells as described previously (27, 55). GST and GST-gp96 were incubated with glutathione CHR-6494 beads (17-5130; GE Healthcare) at 4C for 8 h. The beads were washed 5 times with phosphate-buffered saline (PBS) and then incubated with purified gH/gL/gQ1/gQ2 complex and/or Fc protein at 4C for 8 h. The beads were washed with PBS three times, and the proteins bound to the beads were eluted with elution buffer (C600325; Sangon Biotech) for Coomassie brilliant blue (CBB) staining and immunoblot analysis with appropriate antibodies. Infection inhibition assay. To analyze the inhibitory effect of an anti-gp96 antibody on HHV-6 infection, MT4 and HSB-2 cells were pretreated with the anti-gp96 polyclonal antibody or control serum at 37C for 30? min and then inoculated with cell-free HHV-6A and HHV-6B, respectively. After viral adsorption for 60?min, the inoculum was.