Volume 7 issue 1 January 2006
TARGETING OF PROTEINS OF THE STRIATIN FAMILY TO DENDRITIC SPINES: ROLE OF THE COILED-COIL DOMAIN.
Stéphane Gaillard, Yannick Bailly, Marion Benoist, Tatiana Rakitina, Jean-Pierre Kessler, Laure Fronzaroli-Molinières, Bénédicte Dargent and Francis Castets

Supplementary data
SG2NA still binds caveolin, phocein and calmodulin when its coiled-coil region is deleted.

Legend

A : Caveolin interaction.
GST and GST-caveolin coated beads were incubated with SG2NA-1-465 (Input) and SG2NA-1-465 ? CC (Input) and analyzed by Western-blotting with anti-SG2NA antibodies (A1). Ponceau red staining of nitrocellulose membrane shows that an equivalent amount of GST and GST-caveolin were used (A2). Molecular weights are indicated (MW).
B : Phocein interaction.
GST and GST-phocein coated beads were incubated with COS-7 cell lysates expressing either HA-SG2NAß or HA-SG2NAß ?84-137. Bound proteins were analyzed by Western-blotting with anti-HA antibodies.
C : Calmodulin interaction .
COS-7 cell lysates expressing either HA-SG2NAß or HA-SG2NAß ?84-137 were incubated with calmodulin-agarose in the presence of Ca²⁺. Calmodulin-agarose beads were washed and bound proteins were recovered in the presence of EDTA (bound). An aliquot of COS-7 lysate (Input), unretained (flow-through, FT) and bound fractions were run on 8% SDS-PAGE and recombinant SG2NA was revealed by Western-blotting with anti-HA antibody.

Methods

Recombinant SG2NA fragments, GST, GST-phocein and GST-caveolin-1 were produced in E. coli and purified as described previously ( Baillat et al. 2002, J. Biol. Chem.; 277:18961-6; Gaillard et al. 2001, FEBS Lett.;508:49-52).
GST-pull-down. Caveolin : 100 pmol of purified fragment corresponding to the first 465 amino-acids of SG2NAß (SG2NA-1-465), and the same fragment devoid of the residues 84 to 137 (SG2NA-1-465 ? CC), were incubated overnight at room temperature with either GST-caveolin-1 (300 pmol) or GST (300 pmol) bound to Glutathione-Sepharose 4B beads (Amersham Bio-science) in TBS-CHAPS buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 7.5 mM CHAPS). After extensive washing retained proteins were separated on 11% SDS-PAGE and transferred onto nitrocellulose. Proteins were colored by Ponceau red and SG2NA was revealed with an affinity-purified anti-SG2NA antibody. Phocein: COS-7 cells expressing either HA-SG2NAß or HA-SG2NAß ?84-137 were lysed in RIPA buffer. The lysates were incubated overnight at 4° with GST-phocein or GST bound to Glutathione-Sepharose 4B beads. After extensive washing, proteins retained on beads were analyzed on 8% SDS-PAGE and revealed by Western-blotting with anti-HA antibody.
Calmodulin binding. COS-7 cells expressing either HA-SG2NAß or HA-SG2NAß ?84-137 were lysed in TBS-calcium (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM Mg²⁺, 1mM Ca²⁺) and then incubated for 2H at 4°C with calmodulin-agarose (Sigma). After several washes, bound proteins were eluted in TBS-EDTA (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA). Recombinant proteins were revealed by Western-blotting with anti-HA antibody.

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