Figure 4.

SRFBP1 Colocalizes with CD81 without Affecting Entry Factor Surface Expression

(A) SRFBP1 partially colocalizes with CD81 and the membrane marker WGA but only weakly with CLDN1, OCLN, SR-BI, and GLUT4. Huh-7.5 cells were stained with Alexa-conjugated membrane marker WGA (panel 3) for 1 min or left unstained (panels 1, 2, and 4), fixed, permeabilized, and stained for SRFBP1 and the indicated protein. Nuclei were stained with DAPI. Colocalization across a section (yellow line in panel 1) is depicted above the respective image. Representative confocal images; insert 2.2-fold magnification; scale bars 10 μm.

(B) Pearson’s correlation coefficient for SRFBP1 and the indicated cellular protein or the membrane marker WGA calculated by intensity correlation analysis. Each symbol represents an individual frame; horizontal lines indicate the mean ± SEM.

(C) SRFBP1-silenced cells (siRNA 394) express CD81, CLDN1, and SR-BI at the plasma membrane. Surface expression of CD81, CLDN1, and SR-BI on Huh-7.5 cells was analyzed 48 hpt with the indicated siRNAs. Cells were stained with antibodies against HCV entry factors followed by flow cytometric analysis of 10,000 cells per sample. For quantification and additional controls, see Figure S4. Control is directly conjugated isotype antibody (histogram 1) or secondary antibody only (histograms 2 and 3).

(D) OCLN expression levels are stable after SRFBP1 silencing (siRNA 394). Immunoblot analysis of OCLN (red) and SRFBP1 (green) after siRNA mediated silencing for 48 hr is shown. Actin served as loading control. Data are representative of at least three independent experiments. See also Figure S4.