Fig. 2.

Rottlerin inhibits nSMase2 translocation in response to TNF and PMA. A, total lysate was prepared from A549 cells and immunoblotted for novel PKCs as described under Materials and Methods. B and C, A549 cells (20–25 × 10³) were seeded in 35-mm confocal dishes and 48 h later were transiently transfected with 3′V5-tagged nSMase2 (0.25 μg/dish) for 12 h, media were changed and cells were allowed to grow for 6 to 12 h before stimulation as indicated. Cells were preincubated with rottlerin (10 μM) for 1 h before stimulation as indicated. Cells were fixed and stained with anti-V5 (green) and anti-giantin (red) as described under Materials and Methods. B, effect of rottlerin on PMA-stimulated nSMase2 translocation. After preincubation, cells were stimulated with PMA (100 nM) for 30 min. C, effect of rotterlin on TNF-stimulated nSMase2 translocation. After preincubation, cells were stimulated with TNF (50 ng/ml) for 30 min. Pictures are representative of more than five fields taken from at least four independent experiments.