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. Author manuscript; available in PMC: 2017 Jul 1.
Published in final edited form as: J Neurochem. 2016 May 16;138(1):60–73. doi: 10.1111/jnc.13635

Figure 2. Chemical inhibition of MLCK does not affect vesicle reacidification.

Figure 2

A, Sample experiments on vesicle reacidification at boutons incubated with 0.05% DMSO (Nbouton = 34) or 5 μM wortmannin (Nbouton = 24) for 20 min at 37 °C before imaging. SypHy fluorescence, which was imaged every 500 ms, has been normalized to the amplitude of increase evoked by the 20 Hz stimulation (ΔF20Hz). Boutons were perfused constantly with either the standard solution of pH 7.4 or the solution of pH 5.5 before and after stimulation (started at 30 s, ∼110 s). By fitting the fluorescence decay during the quench (green line) between ∼35 – 60 s after the cessation of stimulation, we measured the time constant of vesicle reacidification as 7.5 s for DMSO and 6.4 s for wortmannin, respectively. B, Averaged changes of SypHy fluorescence to pH 5.5 in the time window indicated by gray frames in A. Boutons were pre-incubated with DMSO (Nexp = 5) or wortmannin (Nexp = 7). The similar fluorescence decay after rapid quenching of surface fluorescence suggests that wortmannin does not alter vesicle reacidification. C, Averaged time constants of vesicle reacidification from boutons incubated with DMSO and wortmannin.