Figure 7.
Phosphorylation is required for Numb localization to the basolateral membrane in response to cell polarization. Polarized MDCK cells transfected with GFP-Numb (A) or GFP-Numb2A (B) were switched to low-calcium media (0 Ca2+) for 20 h followed by Ca2+ re-addition for 24 h to allow for repolarization. Changes in the localization of GFP-Numb, aPKC, and ZO-1 were determined by immunostaining as in Figure 6 (A, B). (C) Colocalization of Numb with the apical membrane marker gp135 in a calcium switch experiment was determined as in Figure 6. Merged images of GFP-Numb or GFP-Numb2A (green), and gp135 (red) staining in optical sections taken in the Z-axis are shown. Colocalization of GFP-Numb (left panels) and GFP-Numb2A (right panels) with gp135 after calcium readdition was quantified using the colocalization module of Improvision Volocity 3.7 software program. Values are given in the text. Size bars indicate 10 μm.