Enzymatic activity of CD13 is needed for optimal organization of membrane proteins. (A) CD13 enzyme activity was determined in vector-transfected EOMA cells (control) and in cells transfected with expression plasmids containing wild-type human CD13 (CD13 [WT]), CD13 point mutants His³⁸⁸Ala (H388A) or His³⁹²Ala (H392A). (B) Total levels of flotillin-1 protein are equivalent in cell lysates from all 4 EOMA lines by Western blot analysis. (C) Triton-solubilized cell lysates from control, wild-type, or mutant-transfected cells were analyzed by sucrose gradient separation and fractions assayed for flotillin-1 by Western blot analysis. High-density fractions (fractions 1-5) represent soluble proteins and light fractions (fractions 9-12) contain proteins complexed with lipids. (D) Ratios of flotillin-1 detected in light and heavy sucrose gradient fractions were calculated using densitometric quantification of Western blots of the 4 EOMA lines. Data are shown as means (± SD, n = 3).