FIG. 2.

Partial colocalization of TBC1D20 and HCV NS5A. (A) Plasmids encoding GFP-fused TBC1D20 and NS5A fused to Ds-Red were cotransfected into Huh7 cells. The cells were fixed 24 h after transfection. (B) Colocalization of TBC1D20 and NS5A in full-length replicon cells. A plasmid encoding GFP-fused TBC1D20 was transfected into FLRP1 cells. The cells were fixed after 24 h and stained with anti-NS5A primary and Alexa 594 secondary antibodies. (C) Colocalization of TBC1D20 and NS5A in replicon cells increases under low-temperature transport inhibition. A plasmid encoding GFP-fused TBC1D20 was transfected into cells expressing the Bart-HA replicons (a replication-competent subgenomic replicon with an HA tag inserted into the C-terminal region of NS5A). Twenty-four hours after transfection, the cells were incubated for 90 min at 16°C, followed by immediate fixation. The cells were stained with anti-HA primary and Alexa 594 secondary antibodies. (A to C) Experiments were visualized using a confocal microscope. On the right are merged images of the corresponding left-hand and middle single-channel fluorescent images. Prominent colocalizations are marked with white arrows. The insets in the left lower corners are enlargements of selected colocalized regions (indicated by arrowheads). (D) Representative fluorescence intensities showing colocalization in a cell with low levels of TBC1D20 expression. The left-hand image indicates the areas of colocalization through which line scan intensities were analyzed (note that these are the same colocalizations indicated by the box with an asterisk in the right-hand image of panel B). The middle image indicates the levels of lines scanned (x and y) to record fluorescence intensities in each of the red and green channels. The right-hand image indicates the actual lines scanned (white lines) with the corresponding fluorescence intensity recordings, which are indicative of colocalization.