Fig. 4.

ERα36 neutralizing Ab attenuates E2-induced SphK1 activation and S1P formation and secretion. MDA-MB-231 cells were pretreated without or with anti-ERα36 neutralizing Ab and then stimulated with vehicle or with E2 (100 nM) for the indicated times. A: Proteins in cell lysates were separated by SDS-PAGE and immunoblotted with the indicated Abs. p-SphK1 and p-Akt were quantified by densitometry, and data are expressed as relative densities normalized to GAPDH. Cellular S1P and S1P released into the medium (B) and cellular S1P and dihydro-S1P (DHS1P) (C) released into the medium during 30 min secretion assays were measured by LC/ESI/MS/MS. Data are mean ± SD. * P ≤ 0.05; # P < 0.001 (compared with vehicle).