PMID- 23348044
OWN - NLM
STAT- MEDLINE
DCOM- 20130816
LR  - 20201209
IS  - 1872-678X (Electronic)
IS  - 0165-0270 (Linking)
VI  - 214
IP  - 1
DP  - 2013 Mar 30
TI  - A HaloTag(R) method for assessing the retrograde axonal transport of the p75 
      neurotrophin receptor and other proteins in compartmented cultures of rat 
      sympathetic neurons.
PG  - 91-104
LID - S0165-0270(13)00024-1 [pii]
LID - 10.1016/j.jneumeth.2013.01.006 [doi]
AB  - We have adapted HaloTag(R) (HT) technology for use in compartmented cultures of rat 
      sympathetic neurons in order to provide a technique that can be broadly applied 
      to studies of the retrograde transport of molecules that play roles in 
      neurotrophin signaling. Transfected neurons expressing HT protein alone, HT 
      protein fused to the p75 neurotrophin receptor (p75NTR) or HT protein fused to 
      tubulin alpha-1B were maintained in compartmented cultures in which cell bodies and 
      proximal axons of rat sympathetic neurons reside in proximal compartments and 
      their distal axons extend into distal compartments. HT ligand containing a 
      fluorescent tetramethylrhodamine (TMR) label was applied either in the distal 
      compartments or the proximal compartments, and the transport of labeled proteins 
      was assayed by gel fluorescence imaging and TMR immunoblot. HT protein expressed 
      alone displayed little or no retrograde transport. HT protein fused to either the 
      intracellular C-terminus or the extracellular N-terminus of p75NTR was 
      retrogradely transported. The retrograde transport of p75NTR was augmented when 
      the distal axons were provided with nerve growth factor (NGF), brain-derived 
      neurotrophic factor (BDNF) or antibodies to BDNF. The anterograde transport of HT 
      protein fused to the N-terminus of tubulin alpha-1B was also demonstrated. We 
      conclude that retrograde transport of HT fusion proteins provides a powerful and 
      novel approach in studies of axonal transport.
CI  - Copyright (c) 2013 Elsevier B.V. All rights reserved.
FAU - Mok, Sue-Ann
AU  - Mok SA
AD  - Department of Cell Biology, Faculty of Medicine and Dentistry, University of 
      Alberta, Edmonton, Alberta T6G 2H7, Canada.
FAU - Lund, Karen
AU  - Lund K
FAU - Lapointe, Paul
AU  - Lapointe P
FAU - Campenot, Robert B
AU  - Campenot RB
LA  - eng
GR  - Canadian Institutes of Health Research/Canada
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20130121
PL  - Netherlands
TA  - J Neurosci Methods
JT  - Journal of neuroscience methods
JID - 7905558
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Ligands)
RN  - 0 (Nerve Tissue Proteins)
RN  - 0 (Receptors, Growth Factor)
RN  - 0 (Receptors, Nerve Growth Factor)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (Rhodamines)
RN  - 0 (Tubulin)
RN  - 0 (tubulin alpha-1B, rat)
RN  - 136958-07-1 (Ngfr protein, rat)
RN  - 62669-72-1 (tetramethylrhodamine)
RN  - EC 3.- (Hydrolases)
RN  - EC 3.8.1.5 (haloalkane dehalogenase)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - *Axonal Transport/physiology
MH  - Base Sequence
MH  - Cell Culture Techniques/instrumentation/methods
MH  - Cells, Cultured/chemistry/metabolism
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Electroporation
MH  - Fluorescent Dyes/analysis
MH  - Fluorometry
MH  - Hydrolases/*analysis/genetics
MH  - Immunoblotting
MH  - Ligands
MH  - Molecular Sequence Data
MH  - Nerve Tissue Proteins/analysis/*metabolism
MH  - Neurons/*physiology/ultrastructure
MH  - Rats
MH  - Rats, Sprague-Dawley
MH  - Receptors, Growth Factor
MH  - Receptors, Nerve Growth Factor/analysis/*metabolism
MH  - Recombinant Fusion Proteins/analysis/genetics/metabolism
MH  - Rhodamines/analysis
MH  - Superior Cervical Ganglion/cytology
MH  - Transfection
MH  - Tubulin/analysis/genetics/*metabolism
EDAT- 2013/01/26 06:00
MHDA- 2013/08/21 06:00
CRDT- 2013/01/26 06:00
PHST- 2012/05/17 00:00 [received]
PHST- 2012/10/19 00:00 [revised]
PHST- 2013/01/08 00:00 [accepted]
PHST- 2013/01/26 06:00 [entrez]
PHST- 2013/01/26 06:00 [pubmed]
PHST- 2013/08/21 06:00 [medline]
AID - S0165-0270(13)00024-1 [pii]
AID - 10.1016/j.jneumeth.2013.01.006 [doi]
PST - ppublish
SO  - J Neurosci Methods. 2013 Mar 30;214(1):91-104. doi: 
      10.1016/j.jneumeth.2013.01.006. Epub 2013 Jan 21.