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, Three days after transfection, cells were lysed and cGAS protein expression was assessed as described in the Experimental Procedures section using two rabbit polyclonal anti-cGAS antibodies (HPA031700, Sigma & NBP1-86761, Novus Biologicals). Given the proximity of the bands, another western blot was run in parallel for the detection of ?-actin as a control

. Detection, DNA was extracted using QiaAMP DNA MiniKit (Qiagen) following manufacturer's instructions. The presence of HBV DNA was confirmed by PCR using the following primers (expected band size: 148 bp) (1) : forward primer 5'CACCTCGCCTAATCATC-3', reverse primer 5'-GGAAAGAAGTCAGAAGGCA-3'. For qPCR quantification of HBV DNA, The presence of HBV DNA was confirmed by PCR and quantified by qPCR using the following primers and probe (1) : forward primer 5'CACCTCGCCTAATCATC-3

, Cells were then fixed with 2% paraformaldehyde for 20 minutes at room temperature. NTCP expression was then quantified by flow cytometry using MacsQuant instrument (Miltenyi), Detection of NTCP expression by flow cytometry. HepG2 cells

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J. Lucifora, A. Salvetti, X. Marniquet, L. Mailly, B. Testoni et al., Detection of the hepatitis B virus (HBV) covalently-closed-circular DNA (cccDNA) in mice transduced with a recombinant AAV-HBV vector, Antiviral Res, vol.145, pp.14-19, 2017.
URL : https://hal.archives-ouvertes.fr/hal-01953660

J. W. Schoggins, D. A. Macduff, N. Imanaka, M. D. Gainey, B. Shrestha et al., Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity, Nature, vol.505, pp.691-695, 2014.