ASMase activity was measured by radioenzymatic assay using [14C-methylcholine] sphingomyelin as substrate, as described with minor modifications . Briefly, following treatment with different conditions, cells were washed with ice cold PBS and lysed in PBS containing 0.2% Triton X-100. 2.5 ug cell lysate were incubated with a total 9.5 nmol sphingomyelin substrate mixture including 0.026 μCi[14C-methylcholine] sphingomyelin in reaction buffer (250 mM sodium acetate, pH 5.0 supplemented with 0.1% Triton X-100 and 1 mM EDTA) at 37° C for 2 h. Reactions were terminated by adding 125 ul of mixture of CHCl3:MeOH:HCl, 100:100:1 v/v/v, and the upper phase was removed and quantified by a Beckman Packard 2200 CA Tricarb scintillation counter.