Fig. 5.

EMK1 promotes “hepatic-type” luminal targeting in nonpolarized cells. Contact-naïve control and EMK1-MDCK monolayers were generated as in Fig. 4. (A) Untransduced cells were incubated with antibodies against the ectodomains of gp135 (green) for 1 h at 37°C. Cell-associated antibodies were detected after fixation and permeabilization with secondary antibodies (ab-uptake). In parallel, samples were fixed, permeabilized, and labeled for total gp135 (immunofluorescence). Shown are 40× wide-field images. (Right) Quantification: Total cell-associated gp135 fluorescence in 30 individual control cells was determined in two separate experiments where gp135 antibodies were added to live cells (striped bars) or after fixation and permeabilization (filled bars). The difference between “total” (filled bars) and “live”(striped bars) fluorescence is statistically significant, P < 0.005. (Schemes in Upper and Lower Left) gp135 immunofluorescence (green, Upper) and gp135 antibody binding and uptake (green and red arrows, Lower) in nonpolarized cells. (B) Cells transduced with an adenovirus encoding LDLR were incubated live with antibodies against the ectodomain of gp135 (green) and LDLR (red) as described in A. Shown are confocal x-y sections. (Bars, 10 μm.) [Image gp135 in A is reprinted from ref. 42 with permission from Elsevier (Copyright 2003, Elsevier).]