FIGURE 4. [³H]Palmitate is incorporated into CD44 by acylation of membrane-proximal cysteines.

A, [³H]palmitate incorporation (250 µCi/ml, 2 h at 37 °C) into Flp-293 stable transfectants of CD44wt, pretreated with or without 50 µm 2-BP (lanes 1 and 2, respectively). [³H]Palmitate incorporation into Flp-293 stable transfectants of Hyal-2 followed by immunoprecipitation using anti-V5 monoclonal antibody is shown in lane 3. In B, the extent of [³H]palmitate incorporation in parental Flp-293 cells (lane 1) or Flp-293 cells expressing the wild-type CD44wt (lane 2) were compared with the Flp-293 expressing the cysteine mutants CD44-C286A (lane 3). Lanes 4 and 5 represent a Western blot (detection using an anti-V5 monoclonal antibody) of equivalent aliquots of lysates shown in lanes 2 and 3, respectively. In C, incorporation of [³H]palmitate into wild-type CD44wt (lane 1), CD44-C286,295A (lane 2), and CD44-C286A (lane 3) were compared side-by-side. Lanes 4–6 represent a Western blot (detection using an anti-V5 monoclonal antibody) of equivalent aliquots of lysates shown in lanes 1–3, respectively. Changes in [³H]palmitate incorporation were quantified by digital scanning of x-ray films, and the resultant data are expressed as the average ± S.D. (n = 3) change in pixel density relative to control CD44 wild-type incorporation.