Plasma membrane proteins are modulated after vaccinia virus-infection

HFFF-TERTs or HeLa cells were mock-treated or infected with VACV at MOI 5 in DMEM supplemented with 2% FCS, 1% P/S. Cells were harvested at 15-18 hpi using accutase solution following the manufacturer’s instructions (Sigma-Aldrich). Validation of PM protein expression was performed with ~2x10^5 cells /sample, stained with Zombie NIR Fixable Viability Kit (Biolegend) in combination with the following antibodies:

MHC-I (W6/32, kindly provided by Dr L. Boyle), MICA (2C10, Santa Cruz Biotechnology, Santa Cruz, CA, USA), EPHB4 (rea923, Miltenyi Biotech, Bergisch Gladbach, Germany), FAS(CD95) (DX2, Miltenyi Biotech), EGFR (528, Santa Cruz), HLA-B/C (4E, kindly provided by Dr L. Boyle), HLA-C/E (DT9, kindly provided by Dr L. Boyle), ULBP3 (166510, R&D systems), B7-H6 (875001, R&D systems, Minneapolis, U.S.A), ULBP-2/5/6 (65903, R&D systems), Plexin-B1 (rea728, Miltenyi Biotech), MICB (236511, R&D systems), TNFSRF10D (trail-R4) (104918, R&D systems), CD155 (SKII.4, Santa Cruz), isotype mouse Igg2b (Santa Cruz), isotype mouse IgG2a (Santa Cruz), isotype mouse IgG1 (Santa Cruz). Samples stained with unconjugated primary antibodies were subsequently probed with PE-conjugated Goat anti-mouse IgG (BioLegend).

Cells were fixed using Fixation/Permeabilization Solution Kit according to the manufacturer’s instructions (BD Biosciences). Samples were analysed by flow cytometry using an Invitrogen Attune NxT cytometer and FlowJo software.