Figure 2.
I2PP2A preferentially binds endogenous C18-ceramide in cells. A) Generation of biotin-labeled C16- or C18-ceramides in A549 cells was induced via the sphingosine recycling pathway. B) Generation of C16- or C18-ceramides in A549 cells was confirmed using B-Sph and 3H-labeled palmitoyl- or stearoyl-CoA (left and right panels) in the absence or presence of FB1or MYR as described in Materials and Methods. C) After forced generation of B-C16- or B-C18-Cer in cells (lanes 2 and 3), the I2PP2A-ceramide interaction in the absence or presence of MYR or FB1 (lanes 5, 8 and 6, 9, respectively) was detected by Western blotting. D) Cells were pretreated with exogenous C6-Pyr-Cer, C16-Pyr-Cer, or C18-Pyr-Cer before treatment with B-C6-Cer (each 10 μM). The competition of the exogenous ceramides with B-C6-Cer in I2PP2A binding (lanes 2–4, respectively) compared with that in controls (lane 1) was then determined as described above.