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. Author manuscript; available in PMC: 2013 Jul 19.
Published in final edited form as: Biochem J. 2011 Apr 15;435(2):381–390. doi: 10.1042/BJ20101752

Figure 4. Stable overexpression of nSMase1 and nSMase2, but not nSMase3, increases in vitro activity of HEK-293 cells independently of assay conditions.

Figure 4

HEK-293 cells stably expressing vector (Vec), nSMase1 (NSM1 or N1), nSMase2 (NSM2 or N2) or nSMase3 (NSM3 or N3) were plated for 24 h. (A) N-SMase activity was assayed by the standard assay in vitro as described in the Materials and methods section. Results are mean ± S.E.M. N-SMase activity (*P < 0.05, n = 4). (B) N-SMase activity was assayed according to the method described by Krut et al. [5] utilizing either labelled SM (hot; light-grey bars) or both labelled and unlabeled SM (hot and cold; dark-grey bars). Results are mean ± S.E.M. N-SMase activity (*P < 0.05, n = 4). (C) Cell lysates were analysed for nSMase1, nSMase2 or nSMase3 expression by immunoblotting (IB) for V5. Blots shown are representative of at least five immunoblots. Molecular-mass markers (in kDa) are shown on the right.