The concentrations and distributions of three C-terminal variants of the GLT1 (EAAT2; slc1a2) glutamate transporter protein in rat brain tissue suggest differential regulation

Holmseth, Silvia; Scott, H. L.; Real, Katia; Lehre, Knut P.; Leergaard, Trygve B.; Bjaalie, Jan G.; Danbolt, Niels Christian

doi:10.1016/j.neuroscience.2009.03.048

Cited by 120 publications

(141 citation statements)

References 65 publications

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“…We hope that sharing our personal experiences will sensitize others to early warning signs and thereby facilitate navigation around some of the difficult situations we have found ourselves in, where a lot of hard work and effort has turned out to have been wasted. Li et al, 2012), was subjected to Western blotting with the anti-B563 antibody (Ab#355; Holmseth et al, 2009) to the extreme C-terminus (residues 563-573) of rat EAAT2 and with the anti-B146 antibodies (Cat. No.…”

Section: Discussionmentioning

confidence: 99%

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Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Danbolt

Zhou

Furness

et al. 2016

Glia

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Immunocytochemistry and Western blotting are still major methods for protein localization, but they rely on the specificity of the antibodies. Validation of antibody specificity remains challenging mostly because ideal negative controls are often unavailable. Further, immunochemical labeling patterns are also influenced by a number of other factors such as postmortem changes, fixation procedures and blocking agents as well as the general assay conditions (e.g., buffers, temperature, etc.). Western blotting similarly depends on tissue collection and sample preparation as well as the electrophoretic separation, transfer to blotting membranes and the immunochemical probing of immobilized molecules. Publication of inaccurate information on protein distribution has downstream consequences for other researchers because the interpretation of physiological and pharmacological observations depends on information on where ion channels, receptors, enzymes or transporters are located. Despite numerous reports, some of which are strongly worded, erroneous localization data are being published. Here we describe the extent of the problem and illustrate the nature of the pitfalls with examples from studies of neurotransmitter transporters. We explain the importance of supplementing immunochemical observations with other measurements (e.g., mRNA levels and distribution, protein activity, mass spectrometry, electrophysiological recordings, etc.) and why quantitative considerations are integral parts of the quality control. Further, we propose a practical strategy for researchers who plan to embark on a localization study. We also share our thoughts about guidelines for quality control. GLIA 2016;64:2045–2064

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Section: Discussionmentioning

confidence: 99%

“…was detected with anti-EAAT2a antibodies (Ab#355: 0.1 µg/ml) followed by HRPconjugated secondary antibodies. This figure is based on materials produced previously (Holmseth et al, 2009). Figure 7.…”

Section: Discussionmentioning

confidence: 99%

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Danbolt

Zhou

Furness

et al. 2016

Glia

View full text Add to dashboard Cite

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“…The supernatants from this last centrifugation are hereafter referred to as "pellet." The pellets and the supernatants were then subjected to SDS-PAGE and immunoblotted as described (45).…”

Section: Methodsmentioning

confidence: 99%

Deletion of the γ-Aminobutyric Acid Transporter 2 (GAT2 and SLC6A13) Gene in Mice Leads to Changes in Liver and Brain Taurine Contents

Zhou

Holmseth

Guo

et al. 2012

Journal of Biological Chemistry

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Background:The physiological roles of GABA transporter 2 (GAT2) are unknown. Results: Deletion of the GAT2 gene reduced liver taurine levels but increased brain levels. Conclusion: GAT2 is unimportant for inactivation of neurotransmitter GABA. Instead, GAT2 is a major taurine transporter in hepatocytes and an efflux transporter at the blood-brain barrier. Significance: GAT2 knockout mice will be crucial for uncovering the roles of GAT2.

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“…The regional distribution of GLT1a and GLT1b in rat brain is very similar, and both variants are primarily found in astrocytes. Neuronal expression of both variants has been observed (15)(16)(17)(18)(19)(20), although other research groups failed to detect GLT1b expression in neurons (15,21). Notably, induction of neuronal GLT1b expression occurs in association with amyotrophic lateral sclerosis and hypoxia, which may be a compensatory reaction to the loss of glial GLT1a that is associated with these pathologies (18,22).…”

mentioning

confidence: 99%

“…GLT1a (Slc2a1, transcript variant 1 in GenBank TM ) is the predominant variant, whereas GLT1b and GLT1c are expressed at lower levels (13,14). In rat hippocampus, GLT1a represents ϳ90% of total GLT1 protein, whereas GLT1b and GLT1c represent ϳ6 and ϳ1%, respectively (15). The regional distribution of GLT1a and GLT1b in rat brain is very similar, and both variants are primarily found in astrocytes.…”

mentioning

confidence: 99%

Functional Modulation of the Glutamate Transporter Variant GLT1b by the PDZ Domain Protein PICK1

Søgaard

Borre

Braunstein

et al. 2013

Journal of Biological Chemistry

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Background:The scaffolding protein PICK1 (protein interacting with C kinase 1) interacts specifically with the glutamate transporter GLT1b. Results: Co-expression of PICK1 and GLT1b increases membrane leak current and alters GLT1b glutamate transport activity. Conclusion:The interaction between PICK1 and GLT1b has functional consequences on transporter kinetics. Significance: Pathology-related neuronal GLT1b expression may lead to PICK1-dependent changes in neuronal excitability.

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The concentrations and distributions of three C-terminal variants of the GLT1 (EAAT2; slc1a2) glutamate transporter protein in rat brain tissue suggest differential regulation

Cited by 120 publications

References 65 publications

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Deletion of the γ-Aminobutyric Acid Transporter 2 (GAT2 and SLC6A13) Gene in Mice Leads to Changes in Liver and Brain Taurine Contents

Functional Modulation of the Glutamate Transporter Variant GLT1b by the PDZ Domain Protein PICK1

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