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Cilia pp 15-33 | Cite as

Targeting of ASH Domain-Containing Proteins to the Centrosome

  • Patricia Verdier
  • Stine K. Morthorst
  • Lotte B. PedersenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1454)

Abstract

A growing number of studies have used new generation technologies to characterize the protein constituents of cilia and centrosomes. This has led to the identification of a vast number of candidate ciliary or centrosomal proteins, whose subcellular localization needs to be investigated and validated. Here, we describe a simple and inexpensive method for analyzing the subcellular localization of candidate cilium- or centrosome-associated proteins, and we illustrate the utility as well as the pitfalls of this method by applying it to a group of ASH (ASPM, SPD-2, Hydin) domain-containing proteins, previously predicted to be cilia- or centrosome-associated proteins based on bioinformatic analyses. By generating plasmids coding for epitope-tagged full-length (FL) or truncated versions of the ASH domain-containing proteins TRAPPC8, TRAPPC13, NPHP4, and DLEC1, followed by expression and quantitative immunofluorescence microscopy (IFM) analysis in cultured human telomerase-immortalized retinal pigmented epithelial (hTERT-RPE1) cells, we could confirm that TRAPPC13 and NPHP4 are highly enriched at the base of primary cilia, whereas DLEC1 seems to associate specifically with motile cilia. Results for TRAPPC8 were inconclusive since epitope-tagged TRAPPC8 fusion proteins were unstable/degraded in cells, emphasizing the need for combining IFM analysis with western blotting in such studies. The method described should be applicable to other candidate ciliary or centrosomal proteins as well.

Key words

ASH domain Centrosome Cilia TRAPPC8 TRAPPC13 NPHP4 DLEC1 

Abbreviations

ASH

ASPM, SPD-2, Hydin

ATCC

American-type culture collection

DAPI

4′ 6-Diamidino-2-phenylindole, dihydrochloride

DLEC1

Deleted in lung and esophageal cancer 1

DMEM

Dulbecco’s modified Eagle’s medium

DTT

Dithiothreitol

ECL

Enhanced chemiluminescence

EDTA

Ethylenediaminetetraacetic acid

EGFP

Enhanced GFP

FAP

Flagella-associated protein

FL

Full length

GFP

Green fluorescent protein

hTERT-RPE1

Human telomerase-immortalized retinal pigmented epithelial

IFM

Immunofluorescence microscopy

LB

Luria-Bertani

LDS

Loading sample buffer

MFI

Mean fluorescence intensity

MSP

Major sperm protein

NPHP4

Nephronophthisis 4

OCRL

Oculocerebrorenal syndrome of Lowe

PACT

Pericentrin-AKAP450 centrosomal targeting

PBS

Phosphate-buffered saline

RT

Room temperature

siRNA

Small interfering RNA

TBS-T

Tris-buffered saline with Tween-20

TPR

Tetratricopeptide

TRAPP

Transport protein particle

TRAPPC

TRAPP complex

Notes

Acknowledgments

This work was supported by the University of Copenhagen Excellence Programme for Interdisciplinary Research and the Danish Council for Independent Research (1331-00254). S.K.M. was partially supported by a Ph.D. fellowship from the Department of Biology, University of Copenhagen. We thank Sophie Saunier for the gift of human NPHP4 cDNA, Søren L. Johansen for technical assistance, Kenneth B. Schou for assistance with DLEC1 and NPHP4 PCR and cloning, and Louise Lindbæk for assistance with image analysis.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Patricia Verdier
    • 1
  • Stine K. Morthorst
    • 1
  • Lotte B. Pedersen
    • 1
    Email author
  1. 1.Department of BiologyUniversity of CopenhagenCopenhagen, OEDenmark

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