Shedding of CD44 or syndecan-1 is mediated by KIF5B and KIF3A/KIF3B, but not by KIF1C. (A-G,J-P) Confocal micrographs of macrophages transfected with siRNA against (A,J) control sequence, (B,K) KIF5B, (C,L) KIF3A, (D,M) KIF3B, (E,N) KIF5B and KIF3B, (F,O) KIF1C, or (G,P) MT1-MMP. Cells were fixed, but not permeabilized, and stained with primary anti-CD44 (A-G) or anti–syndecan-1 (J-P) antibody, respectively, recognizing an epitope in the ectodomain, and secondary Cy5-conjugated antibody. White scale bar indicates 10 μm for all images. (H,Q) Fluorescence intensities of surface-localized (H) CD44 or (Q) syndecan-1, based on Cy5 fluorescence. Fluorescence intensities for the control siRNA were set to 100%. For all values, 3 × 30 cells were evaluated. For specific values, see supplemental Table 1. (I,R) ELISA for measuring shedded ectodomains of (I) CD44 or (R) syndecan-1 in culture media of siRNA-treated cells described in panels A through H and J through Q. Data were evaluated using the ratio of OD450 to OD620 to exclude a noncompleted development of the substrate. Experiments were performed twice in triplicates, with values shown as mean ± SD of 1 experiment. For specific values, see supplemental Table 1. *P < .05, **P < .01 compared with control.