Figure 1.
Establishment of SphK1 and SphK2 expressing HEK293 cell lines and validation of the SphK antibodies. (a) Naïve HEK293 cells and separate clones over-expressing FLAG-SphK1 and FLAG-SphK2 were analyzed for expression of SphK1 and SphK2 using anti-FLAG and SphK isoform specific antibodies by western blot analysis, as indicated. GAPDH was included as a loading control. (b) HEK293 cell lysates over-expressing FLAG-SphK1 and FLAG-SphK2 were combined at the indicated ratios and analyzed for expression of SphK1 and SphK2 using anti-FLAG antibodies and simultaneously using both SphK isoform specific antibodies at 1:2000 dilutions by western blot analysis. GAPDH was included as a loading control.