FIG 4.

Regions of 16L2 downstream of the N-terminal putative transmembrane domain become accessible in selectively permeabilized cells. HaCaT cells were infected for 24 h with HPV16 pseudovirus. Cells were fixed and permeabilized using 0.5% Triton X-100 (A, C, E, and G) or 5 μg/ml digitonin (B, D, F, and H) and stained using L2-specific MAbs (K4, K1, and 33L2-1) or an L1-specific MAb (33L1-7). The ER was stained using MAb against the luminal ER marker calnexin (Calnx). Cells were fixed again and permeabilized using 0.5% Triton X-100, followed by treatment with the Click-iT reaction mixture to detect EdU. Note that only the L2-specific MAb K4, but not K1 and 33L2-1 MAbs, fails to detect the L2 protein adjacent to the nucleus, probably residing in the TGN (arrows), under selective permeabilization. (G and H) As a control, we also did not detect 33L1-7 staining following the Click-iT reaction adjacent to the nucleus under selective permeabilization. Colors are coded as indicated by the labels on the figure.