Post-Translational Tubulin Modifications in Human Astrocyte Cultures

Neurochemical Research
V Bleu Knight, Elba E Serrano

Abstract

The cytoskeletal protein tubulin plays an integral role in the functional specialization of many cell types. In the central nervous system, post-translational modifications and the expression of specific tubulin isotypes in neurons have been analyzed in greater detail than in their astrocytic counterparts. In this study, we characterized post-translational specifications of tubulin in human astrocytes using the normal human astrocyte (NHA; Lonza) commercial cell line of fetal origin. Immunocytochemical techniques were implemented in conjunction with confocal microscopy to image class III β-tubulin (βIII-tubulin), acetylated tubulin, and polyglutamylated tubulin using fluorescent antibody probes. Fluorescent probe intensity differences and colocalization were quantitatively assessed with the 'EBImage' package for the statistical programming language R. Colocalization analysis revealed that, although both acetylated tubulin and polyglutamylated tubulin showed a high degree of correlation with βIII-tubulin, the correlation with acetylated tubulin was stronger. Quantification and statistical analysis of fluorescence intensity demonstrated that the fluorescence probe intensity ratio for acetylated tubulin/βIII-tubulin was greater th...Continue Reading

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Methods Mentioned

BETA
acetylation
Protein Assay
electrophoresis
Fluorescence
RNA-seq

Key Resources (RRID) Mentioned

AB_2631274
AB_297929
AB_609894
AB_869991
AB_2631447

Software Mentioned

R
ggplot2
EbImage
Photoshop
ZenLightEdition
ggthemes
Image Lab
Zeiss

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