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. 2015 Jun 15;16(6):13649–13652. doi: 10.3390/ijms160613649

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© 2015 by the authors; licensee MDPI, Basel, Switzerland.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).

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Analysis of sphingomyelin phosphodiesterase 1 (SMPD1) sequence variations by transient transfection studies. The full-length SMPD1 cDNA was cloned into the FLAG-N2 expression vector, and the SMPD1 variants p.V36A, p.P325A, p.A487V and p.G508R were generated by site-directed mutagenesis. The variant cDNAs and the empty FLAG-N2 vector (mock) were transiently transfected into MDCK cells, and acid sphingomyelinase activity was determined from cell lysates (L-ASM activity; upper panel) and supernatants (S-ASM activity; lower panel). Representative results of a typical experiment with three replicates are given as fold increase over the mock-transfected control. Error bars indicate the standard deviation. With the exception of p.P325A, all mutants increased acid sphingomyelinase activity by >fivefold, similarly to SMPD1 wild-type. Methods are described in detail in [5].