Fig 1: ERp57 down-regulation inhibits HBV entry.(A) Intracellular (upper panels) and cell-surface (lower panels) staining of ERp46, ERp57, and ERp72 protein disulfide isomerase (PDI) members. Huh7-NTCP cells were subjected to flow cytometry analysis, in order to evaluate the expression of the indicated PDIs. Cells stained with secondary antibody only (no primary) were used to provide the background of flow cytometry analyses. (B) Hepatitis delta virus (HDV) or (C) hepatitis B virus (HBV) particles were used to infect Huh7-NTCP cells in which the indicated PDIs were down-regulated by lentiviral vectors carrying shRNA (see Figure 7—figure supplement 1 and Figure 7—figure supplement 2). Naive Huh7-NTCP cells were used as controls. Infected cells were grown for 7 days before total intracellular RNA or DNA was purified. The results of HDV RNA and HBV DNA quantification by quantitative reverse transcription PCR (RTqPCR) and quantitative PCR (qPCR), respectively, are expressed after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) RNAs as means ± SD (N = 3) per ml of cell lysates containing 106 cells. (D) Huh7 ‘donor’ cells co-expressing HBV glycoproteins (GPs) and a luciferase marker gene driven by the HIV-1 promoter were co-cultured with Huh7-NTCP-tat ‘indicator’ cells that express HIV Tat protein in which the indicated PDIs were down-regulated by lentiviral vectors carrying shRNA. After 24 hr, the cells were treated at pH 4 or pH 7 for 3 min. The luciferase activity induced by the fusion between donor and indicator cells was measured 24 hr later. Fusion mediated by HBV GPs at pH 7 with naive Huh7-NTCP-tat cells (Ctrl) was taken as 100%. A control plasmid that does not allow GP expression (Empty) was used to determine the background of luciferase expression. The bars represent the means (N = 3). Error bars correspond to standard deviations.Figure 7—source data 1.ERp57 down-regulation inhibits HBV entry.The values correspond to the data expressed in the graphs displayed in Figure 7B–D.
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