Skip to main content
. 2022 Mar 30;14(4):727. doi: 10.3390/v14040727

Figure 2.

Figure 2

Knockdown of IFITM3 and NTCP expression. PHHs as well as NTCP-expressing and non-NTCP-expressing HepG2 and HuH7 cells were transiently transfected with either control or IFITM3 siRNA and incubated for 72 h. (A–C) Cells were lysed using RNA lysis buffer and processed for determination of the gene expression of NTCP and IFITM3 via qRT-PCR. Expression of GAPDH served as an endogenous control, and non-siRNA-transfected cells served as calibrator (set to a value of 1). Data represents means ± SD of two (HepG2 and HuH7) or three (PHH) independent experiments each with duplicate determinations (n = 4 and n = 6, respectively). * Significantly lower mRNA expression compared to control siRNA transfected cells with p < 0.05 according to two-way ANOVA with Sidak multiple comparisons test. (D–F) Cells were lysed using protein lysis buffer and processed for Western blotting with antibodies against FLAG, NTCP, IFITM3, or GAPDH, respectively. Band intensities were quantified by using the Image Lab software and expression of FLAG/NTCP was normalized to GAPDH, respectively. (G) Cells were incubated with NHS-SS-biotin prior to lysis, and pulldown with streptavidin-coupled beads was performed to separate surface proteins. All samples were processed for Western blotting using an anti-FLAG antibody and an anti-GAPDH antibody.