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1.
Figure 1

Figure 1. Expression of full-length Myc- and FLAG-tagged GRIP domain proteins. From: The trans-Golgi network GRIP-domain proteins form ?-helical homodimers.

HeLa cells were transfected with Myc- or FLAG-tagged GCC88, golgin-97, GCC185 or p230, as indicated. Extracts of transfected cells were analysed by immunoblotting with (A) monoclonal anti-Myc antibodies and (B) monoclonal anti-FLAG antibodies, as indicated, using a chemiluminescence detection system.

Michael R. Luke, et al. Biochem J. 2005 June 15;388(Pt 3):835-841.
2.
Figure 2

Figure 2. Localization of Myc- and FLAG-tagged GRIP domain proteins in transfected HeLa cells. From: The trans-Golgi network GRIP-domain proteins form ?-helical homodimers.

HeLa cells were transfected with constructs encoding either Myc- or FLAG-tagged GRIP domain proteins, as indicated, fixed, permeabilized and stained with anti-Myc or anti-FLAG monoclonal antibody followed by FITC goat anti-mouse Ig. Scale bar, 10 μm.

Michael R. Luke, et al. Biochem J. 2005 June 15;388(Pt 3):835-841.
3.
Figure 5

Figure 5. Cross-linking of cytosolic GCC88. From: The trans-Golgi network GRIP-domain proteins form ?-helical homodimers.

Untransfected HeLa cells, or pCI-GCC88 transfected HeLa cells, were extracted and extracts treated with the indicated concentrations (mM) of the cross-linker DTSSP and analysed by non-reducing SDS/PAGE followed by immunoblotting with rabbit anti-GCC88 antibodies or preimmune serum as indicated, using a chemiluminescence detection system.

Michael R. Luke, et al. Biochem J. 2005 June 15;388(Pt 3):835-841.
4.
Figure 3

Figure 3. Co-immunoprecipitation of epitope-tagged GRIP domain proteins. From: The trans-Golgi network GRIP-domain proteins form ?-helical homodimers.

(A) Strategy for determining interactions between GRIP domain proteins. HeLa cells were co-transfected with pairs of Myc- and FLAG-tagged constructs and extracts of transfected cells were immunoprecipitated with anti-Myc antibody. The immunoprecipitate was then blotted with anti-FLAG and anti-Myc antibodies. (B) Co-immunoprecipitation of Myc–golgin-97 with either FLAG–golgin-97 or FLAG–GCC88. (C) HeLa cells were co-transfected with pairs of Myc- and FLAG-tagged constructs, as indicated, and extracts of transfected cells were analysed directly (Total) or immunoprecipitated with anti-Myc antibody (IP). Samples were blotted with anti-FLAG or anti-Myc antibodies, as indicated.

Michael R. Luke, et al. Biochem J. 2005 June 15;388(Pt 3):835-841.
5.
Figure 7

Figure 7. CD spectrum of purified His6-tagged golgin-97. From: The trans-Golgi network GRIP-domain proteins form ?-helical homodimers.

(A) Purified His6-tagged golgin-97 analysed by SDS/PAGE and stained with Coomassie Blue. (B) CD spectrum of purified His6-tagged golgin-97 (0.42 mg/ml). The change in ellipticity, Δε (M−1·cm−1), is plotted as a function of wavelength (nm) for golgin-97 (0.42 mg/ml) dissolved in PBS (open symbols). The data are superimposed with the non-linear best-fit using the program CDSSTR [26], yielding 67% α-helix, 12% β-strand, 9% turn and 12% unordered structure (—) with an r.m.s.d. of 0.277.

Michael R. Luke, et al. Biochem J. 2005 June 15;388(Pt 3):835-841.
6.
Figure 4

Figure 4. The GRIP domain proteins form homo-oligomers. From: The trans-Golgi network GRIP-domain proteins form ?-helical homodimers.

HeLa cell monolayers were co-transfected with (A) Myc–GCC88, (B) Myc–golgin-97, (C) Myc–GCC185, (D) Myc–p230 and FLAG-tagged full-length GRIP domain proteins, as indicated. The cells were harvested and complexes containing the Myc-tagged proteins were immunoprecipitated with anti-Myc antibodies. Samples of total extract (Total) and immunoprecipitate (IP) were analysed by immunoblotting with monoclonal anti-Myc or anti-FLAG antibodies, as indicated, followed by horseradish peroxidase-conjugated anti-mouse Ig antibodies. Immunoblots were developed by a chemiluminescence detection system. Loadings represent approx. 2% of input and 20% of immunoprecipitate.

Michael R. Luke, et al. Biochem J. 2005 June 15;388(Pt 3):835-841.
7.
Figure 6

Figure 6. Dimerization of GCC88 constructs analysed by yeast two-hybrid interactions. From: The trans-Golgi network GRIP-domain proteins form ?-helical homodimers.

The indicated GCC88 constructs were generated as fusions with the DNA-binding protein LexA in pHybLex/Zeo and the GAL4 activation domain in pGAD GH. GCC88 refers to the full-length 775-amino-acid sequence, and the amino acid numbers denote truncated constructs. The pHybLex/Zeo and pGAD constructs were co-transformed into yeast reporter strain L40. Transformants were initially selected on medium lacking leucine and containing zeocin. The interaction between LexA and GAL4 fusions was assessed by HIS3 reporter gene activation as measured by the ability of transformants to grow on medium lacking histamine and by LacZ reporter gene activation using the liquid β-galactosidase assay. β-Galactosidase activity is given in Miller units. Assays were performed in triplicate.

Michael R. Luke, et al. Biochem J. 2005 June 15;388(Pt 3):835-841.

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