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. 2010 Oct 7;6(10):e1001130. doi: 10.1371/journal.ppat.1001130

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Eyre et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) HepG2(N6)+CD81 cells expressing the indicated shRNA constructs were cultured under polarizing conditions (5 d post-confluence) prior to Western analysis of PDZK1 (∼70 kDa) and SR-BI (∼85 kDa) protein levels. β-actin (∼42 kDa) was used as a loading control. PDZK1 and SR-BI protein levels were quantified by densitometry and normalized to those of the loading control β-actin (graphs). Data are means + SEM (n = 3). (B) Cell-surface expression levels of SR-BI. HepG2(N6)+CD81 cells (5 d post-confluence) expressing the indicated shRNA targets were surface-biotinylated prior to detergent lysis, streptavidin-precipitation of biotinylated proteins and Western blotting. SR-BI, PDZK1 and CD81 (∼26 kDa) protein levels in whole cell lysates (WCL) (upper panels) and streptavidin-precipitates (lower panels) are shown. Overexpressed CD81 (with a C-terminal Myc epitope tag) was detected with an anti-C-myc antibody. (C) Confluent HepG2(N6)+CD81 cells expressing the indicated shRNA constructs were infected with HCVpp or VSVpp 72 h prior to measurement of luciferase activity. Data are means + SEM (n = 4).