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. 2006 Aug 15;4(9):e271. doi: 10.1371/journal.pbio.0040271

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© 2006 Tsuriel et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Long-term imaging of axons of neurons expressing GFP:Synapsin I, before and after the addition of cycloheximide (CHX). CHX was added to attain a final concentration of 100 μM 1 h 12 min after the beginning of the experiment.

(B) Mean fluorescence levels of all GFP:Synapsin puncta recorded in this experiment normalized to their mean fluorescence intensities at the beginning of the experiment (five cells, 13 fields of view, 197 synaptic boutons).

(C) A similar experiment in which MG132 was added 2 h 29 min after the beginning of the experiment to attain a final concentration of 20 μM. Fluorescence levels were normalized to fluorescence intensities at the beginning of the experiment (four cells, ten fields of view, 225 synaptic boutons).

(D) Paired FRAP experiments were performed in the same preparations of neurons expressing GFP:Synapsin I, first in the absence and then in the presence of cycloheximide (three experiments, 12 boutons before cycloheximide addition, 11 boutons after cycloheximide addition).

(E) Paired FRAP experiments as in (D) but with MG132 instead of cycloheximide (four experiments, 13 boutons before MG132 addition, 16 boutons after MG132 addition). All experiments in (A–E) were performed in CNQX and AP-5 to avoid the confounding effects of spontaneous activity.

(F) Long-term imaging of dendrites of neurons expressing GFP:ProSAP2, before and after the addition of cycloheximide (at 2 h 36 min after the beginning of the experiment).

(G) Mean fluorescence levels of all GFP:ProSAP2 puncta recorded in this experiment normalized to their mean fluorescence intensities at the beginning of the experiment (five cells, five fields of view, 678 PSDs).

(H) A similar experiment in which MG132 was added 3 h 4 min after the beginning of the experiment (seven cells, eight fields of view, 574 PSDs).

(I) Paired FRAP experiments were performed in neurons expressing GFP:ProSAP2, first in the absence and then in the presence of cycloheximide (three experiments, ten PSDs before cycloheximide addition, eight PSDs after cycloheximide addition).

(J) Paired FRAP experiments as in (I) but with MG132 instead of cycloheximide (three experiments, nine PSDs before MG132 addition, nine PSDs after MG132 addition). Data in FRAP experiments were not corrected for photobleaching during the recovery phase to avoid canceling out general changes in puncta fluorescence related to cycloheximide or MG132 addition. For sake of clarity, only one-sided error bars are shown in (D, E, I, J). Bars (A, F), 10 μm.