PrlR localizes basolaterally in mammary epithelial cells cultured in 2D. (A) Immunofluorescence analysis of PrlR (green) and the apical marker ZO-1 (red) in monolayers of EpH4 cells demonstrated that PrlR was localized mainly to the basolateral surface. (B) Western blot analysis of STAT5 phosphorylation in response to Prl treatment. Monolayers of EpH4 cells on permeable filters were incubated with Prl added to apical or basal surfaces for 10 min. (C) Immunofluorescence analysis of ZO-1 in monolayers of EpH4 cells after 5 mM EGTA treatment. (D) Western blot analysis of STAT5 phosphorylation. Monolayers of EpH4 cells were incubated with EGTA for 30 and 60 min followed by treatment with Prl for 10 min. Bars, 25 μm.