Fig 5. Expression and function of E6^R:E7^G.

The parental TfR RNAi cell line containing RNAi resistant E6^R and E7^G (E6^R:E7^G) was cultured without (tet-) or with (tet+) tetracycline. All analyses are identical to Fig 3. A. Cell density. B. Transcript levels by qRT-PCR. C. Biosynthesis and pull-down of TfR subunits. Note that the E6^R and E7^G ORFs/proteins are the same length/size. All phosphorimages are representative of three independent biological replicates. D. Receptor mediated endocytosis by flow cytometry. E. IFA of fixed permeabilized and non-permeabilized cells, as indicated. Permeable cells (left) were stained with anti-BiP (green), rabbit anti-TfR (red), and DAPI (blue) to detect nucleus and kinetoplast. Arrowheads indicate surface staining along the flagellar membrane. Non-permeable cells (right) were stained with anti-TfR alone. Deconvolved three-channel summed stack projections of representative cells (tet-) or (tet+) are shown. Bar = 4 μm.