The T198I mutation has no effect on sybII localisation or surface fraction. Primary cultures of synaptophysin knockout hippocampal neurons were transfected with sybII-pHluorin and either wild-type mCerulean-tagged human synaptophysin (WT), the T198I mutant (T198I) or empty mCer vector (KO). (A) Representative images of neurons transfected with sybII-pHluorin are displayed. The images have been pseudo-coloured to indicate regions of high intensity in white. Scale bar equivalent to 10 μm. (B) Coefficient of variation (CV) analysis for sybII-pHluorin is displayed for neurons co-expressing either WT (black), T198I (red) or mCer (KO, blue) ± SEM (WT n = 11, T198I n = 14, KO n = 18). ** = p < 0.01, * = p < 0.05, ** = p < 0.01, one-way ANOVA against WT. (C) Surface expression of sybII-pHluorin is displayed as a percentage of total sybII-pHluorin for neurons co-expressing WT (black), T198I (red) and mCer (KO, blue) ± SEM (WT n = 11, T198I n = 14, KO n = 18). *** = p < 0.001, one-way ANOVA against WT.