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. 2011 Jul 25;194(2):277–289. doi: 10.1083/jcb.201102051

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© 2011 Khavandgar et al.

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Figure 2. — Effects of fro mutation on nSMase2 activity and localization. (A and B) Schematic depiction of WT (Smpd3; A) and mutant Smpd3 (mSmpd3; carries fro mutation; B) expression constructs. The red boxes represent the FLAG coding sequence (CMV, cytomegalovirus promoter; IRES, internal ribosome entry site; and pA, SV40 polyadenylation signal). (C) Western blots showing expression of WT and mutant nSMase2 in transfected MCF-7 cells. FLAG-tagged proteins from the transfected cells were detected using an anti-FLAG (top) or an anti–mouse nSMase2 (middle) antibody. UT, untransfected. (D) A mixed micelle assay using ¹⁴C-labeled methyl-sphingomyelin shows that mutated nSMase2 does not have any nSMase activity. Error bars represent standard deviations. (E) Indirect immunofluorescence microscopy analyses showing comparable cell membrane localization of WT and mutated nSMase2 (shown in red) in transfected MCF-7 cells. The green and blue stains represent GFP localization and the nucleus, respectively. **, P < 0.01.