Figure 9.

The polarized cell surface distribution of mMeg is not modified by the presence of the SNX17-binding domain of LRP1. The cell surface distribution of mMeg-1, mMeg-2, and mMeg-3 was determined in MDCK cell lines stably expressing the minimegalins. The cells were grown until forming an impermeable monolayer in Transwell chambers. (A) The cells were then selectively biotinylated at 4°C, and the cell lysates were analyzed via Western blotting. All the minireceptors were localized apically. The membrane was stripped and blotted to detect the endogenous protein E-cadherin, showing correct basolateral localization. (B) Polarized MDCK cells expressing mMeg-1, mMeg-2, and mMeg-3 were grown on Transwell filters to analyze the localization of the minireceptors through immunofluorescence and confocal microscopy. The cells were fixed, permeabilized, and incubated with anti-HA (red), to detect the minireceptors, and mouse anti-E-cadherin (green), to determine the endogenous lateral marker. The minireceptors show a predominant apical localization. Representative x–y plane and x–z and y–z sections are shown. Scale bars, 10 μm.