Coprecipitation experiments with extracts from HEK293 cells expressing different GFP-Cobl-like proteins were essentially done as described before (Qualmann et al., 1999 (link); Schwintzer et al., 2011 (link)). In brief, HEK293 cell lysates were incubated for 3 h at 4°C with purified, recombinant GST-fusion proteins immobilized on glutathione sepharose beads (GenScript). The reactions were washed several times with lysis buffer containing 150 mM NaCl and EDTA-free protease inhibitor Complete. Bound protein complexes were eluted either with 20 mM reduced glutathione, 120 mM NaCl, and 50 mM Tris-HCl, pH 8.0 (30 min RT), or by boiling the beads in 4× SDS sample buffer.
For coprecipitations with CaM, HEK293 cell lysates were prepared in an EGTA-free lysis buffer containing 150 mM NaCl, EDTA-free protease inhibitor cocktail, and 200 µM calpain I inhibitor. Cell lysates were supplemented with 1 mM EGTA or 500 µM CaCl2. After incubation with 25 µl CaM-sepharose 4B (GE Healthcare) for 3 h at 4°C and washing, bound proteins were isolated by boiling in SDS sample buffer.
Lysates, supernatants, and eluates were analyzed by immunoblotting using anti-GST and anti-GFP antibodies, respectively.
For coprecipitations with CaM, HEK293 cell lysates were prepared in an EGTA-free lysis buffer containing 150 mM NaCl, EDTA-free protease inhibitor cocktail, and 200 µM calpain I inhibitor. Cell lysates were supplemented with 1 mM EGTA or 500 µM CaCl2. After incubation with 25 µl CaM-sepharose 4B (GE Healthcare) for 3 h at 4°C and washing, bound proteins were isolated by boiling in SDS sample buffer.
Lysates, supernatants, and eluates were analyzed by immunoblotting using anti-GST and anti-GFP antibodies, respectively.