protein we used 12.5% Tricine–SDS-PAGE (Schagger and von Jagow, 1987).
A rabbit polyclonal antibody against the Ric-8B peptide ELPSDKTTEKDTAL
was ordered from Bethyl Laboratories, Inc. (Montgomery, TX). Recombinant
GST-Ric-8B protein was produced using the pGEX 2T vector (Amersham
Biosciences) containing the Ric-8B cDNA and E. coli BL21cells. For affinity
purification, the antiserum was incubated with recombinant GST-Ric-8B
protein immobilized on nitrocellulose. The bound antibodies were eluted with
100 mM glycine–HCl pH 2.5 for 5 min and immediately neutralized with
1 volume of 1 M Tris–HCl pH 8.0. The affinity purified Ric-8B antibody
(0.15 μg/μl) was used at a 1:50 dilution. The Gαolf and Gγ13 antibodies (Santa
Cruz, sc385 and sc26782) were respectively used at 1:300 and 1:500 dilutions.
Pull-down assay
10
6
HEK293T cells were transfected with constructs for myc-tagged
Gαolf, FLAG-tagged Gβ1 and FLAG-tagged Gγ13. 24 h after transfection
cells were washed twice with PBS and lysed in lysis buffer (20 mM Tris–HCl
pH 7.5, 100 mM NaCl, 5 mM MgCl
2
, 0.1% Triton X-100, 100 mM lactose)
containing 1× protease inhibitor cocktail (Sigma). Total protein content was
estimated by Bradford. 1300 μg of extract was pre-cleared with 200 μlof
Glutathione–Sepharose 4B in lysis buffer for 1 h. A 50 μl sample was saved
and used as input. The pre-cleared extract was aliquoted in two samples
containing 600 μg of protein each. Both samples were incubated for 2 h at 4 °C
under gentle agitation, one of them was added with 20 μg of recombinant GST-
Ric-8B immobilized in Glutathione–Sepharose, the other one (negative
control) was added with an equal volume of 50% Glutathione–Sepharose.
Samples were washed once with lysis buffer and 2 times with 20 mM Tris–
HCl pH 7.5, 100 mM NaCl, 5 mM MgCl
2
, 0.1% Triton X-100, 25 mM lactose.
Recovered proteins were separated using 12%/18% SDS-PAGE followed by
Western blot analysis using anti-FLAG (1:5000) or anti-myc (1:2000).
Immunohistochemistry
Olfactory turbinates from 3 week old mice were dissected and fixed in 4%
paraformaldehyde for 2 h, washed in PBS and equilibrated in 30% sucrose
overnight at 4 °C. The tissue was embedded in Tissue-Tek OCT compound
(Sakura Finetek, Torrance, CA) and 16–20 μm coronal sections were cut on a
cryostat. Sections were air-dried, fixed in 95% ethanol for 1 min, washed 3
times with PBS, incubated in blocking solution (2% normal fetal bovine
serum, 2% albumin and 0.25% Triton X-100 in PBS) for 1 h, and incubated
overnight at 4 °C with the primary antibodies diluted in blocking solution.
Primary Ric-8B antibodies, rabbit anti-Gαolf, goat anti-Gγ13 and rabbit anti-
Gβ1 (Santa Cruz, sc-379) were used, respectively at 1:50, 1:100, 1:50 and
1:50. Sections were washed three times in PBS, and incubated with the
secondary antibodies (1:500, goat Alexa488-anti-rabbit, donkey Alexa488
anti-goat or donkey Alexa594 anti-rabbit, Molecular Probes, Invitrogen) in
blocking solution for 2 h at room temperature, washed (the first washing
solution contained 0.2 μg/ml DAPI) and mounted in 90% glycerol and 1 mg/
ml p-phenylenediamine in PBS pH8.6. The staining was analyzed using a
LSM 510 META confocal laser scanning microscope from Carl Zeiss.
Acknowledgments
We would like to thank Jean-Pierre Montmayeur and Isaías
Glezer for critically reading the manuscript. We are also grateful to
Adriana Matsukuma for help with the acquisition of confocal
microscope images. We would also like to thank Erica Bandeira
and Edson Alves Gomes for technical assistance. This work was
supported by grants from Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP), Conselho Nacional de Desenvol-
vimento Científico e Tecnológico (CNPq) and CAPES.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.mcn.2008.03.006.
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