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. 2018 Dec;121:38–49. doi: 10.1016/j.neuint.2018.10.014

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© 2018 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Fig. 6 — SNAP-25 mRNA and protein expression during hiPSC differentiation to cortical neurons. (A)SNAP-25 mRNA was detected by quantitative PCR and its expression was first observed on d18. SNAP-25 mRNA expression increased gradually during differentiation until d165, when SNAP-25 mRNA was expressed the highest. SNAP-25 mRNA was normalized to the average of HPRT-1, RPL-27 and RPL-30 mRNA levels. SNAP-25 mRNA fold change in each differentiation was related to the highest SNAP-25 mRNA expression, set to 1. For a summary of assigned significances for the measured changes of SNAP-25 mRNA, see Supplementary Table 10. (B) SNAP-25 protein was first detected on d18 by western blot. SNAP-25 protein levels increased gradually from d18 to d75. After d105, SNAP-25 levels plateaued until d135, followed by reduction in the levels of SNAP-25. SNAP-25 levels were correlated to GAPDH and SNAP-25 fold change in each differentiation was related to the highest SNAP-25 expression, set to 1. Representative bands of SNAP-25 and GAPDH are shown. The summary of assigned significances for the measured changes of SNAP-25 protein is shown in Supplementary Table 11. (C and D) IP followed by PRM mass spectrometry was used to detect secreted SNAP-25; SNAP-25tot (shown in C), and SNAP-25aa40 (shown in D). (C) SNAP-25tot represents all soluble forms of SNAP-25 and was first secreted on d25. The secretion of SNAP-25tot increased markedly from d60 to d135, when it was secreted the highest. Thereafter, the secreted concentrations of SNAP-25tot plateaued until d165 followed by decreased secretion. (D) SNAP-25aa40 measures the longer soluble forms (including at least amino acid 32–40). There was a gradual increase of secreted SNAP-25aa40 between d60 and d105, followed by a marked increase from d105 to d120, when it was secreted the most. After d165, a trend towards decreased secretion of SNAP-25aa40 was observed. The summary of assigned significances for the changes of secreted SNAP-25tot and SNAP-25aa40 concentrations are summarized in Supplementary Table 12 and 13, respectively. Bars represents mean±SEM. For intracellular expression of SNAP-25 mRNA and protein, n = 4 (except from d165 to d210 where n = 2) and for secreted SNAP-25, n = 5 (except from d165 to d210 where n = 3).