(A) 2D projections of 3D confocal stack images of Num1-GFP in WT and scs2/22∆ cells. (B) Fraction of cells with indicated number of Num1-GFP patches. , average number of patches per cell (n ≥ 50 cells per strain). (C) Distribution of Num1-GFP patches along the cortex. The position of each patch was projected on the mother-bud axis and normalized to the bud neck. Positive distances indicate that the patch was in the mother cell, whereas negative distances indicate that the patch was in the daughter cell (n = 46 and 16 cells for scs2/22∆ and WT, respectively). (D) Western blots showing Num1-13myc levels in whole cell lysates of indicated strains. (E) Sucrose gradient sedimentation analysis of Num1-13myc in WT and scs2/22∆ strains. Whole cell lysates from each strain were loaded onto 20-60% sucrose gradients, sedimented, and analyzed by Western blot using anti-c-Myc (for Num1-13myc) and anti-Sac1 (for ER) antibodies. Top, representative sedimentation profiles from two independent experiments. Middle, Num1-13myc band intensity plotted against fraction number. Bottom, Western blot showing Num1-13myc in fractions 17 through 21. (F) Deconvolved wide-field images of Num1-GFP and Scs2-mRuby2 in WT cells. Each image is a 2D projection of 11 optical sections spaced 0.5 µm apart. Green and red arrows indicate Num1-GFP patches that do and do not colocalize with Scs2-mRuby2 foci, respectively. B, bud; M, mother. Bottom, histogram of Pearson’s correlation coefficients for the colocalization of Num1-GFP with Scs2-mRuby2 (n = 200 cortical Num1 patches found in either bud or mother cell).