Inclusion bodies from the medium were lysed with 50 mM Tris-HCl, pH 8.5, 10 mM DTT and 8 M urea from which the His6-p53 was prepared.
The collected cells together with the medium were lysed by four freeze thaw cycles, and subjected to centrifugation at 3000 ×g for 30 min at 4°C for cell debris removal.
Cells expressing histidine-tagged EGFR672 998/V924R (~3 litres of medium) were lysed by sonication in 100 ml of lysis buffer [20 mM Tris/HCl (pH 8.0), containing 500 mM NaCl, 5 mM 2-mercaptoethanol and protease inhibitor cocktail (Roche)].
Sf9 cells expressing histidine-tagged Ror2452 753 (~8 litres of medium) were lysed by sonication in 150 ml of lysis buffer, composed of 20 mM NaKPO4 (pH 8.0), containing 200 mM NaCl, 10 mM 2-mercaptoethanol, 1 mM PMSF, 10 μM benzamidine, 2.3 μM leupeptin, 2 μM aprotinin and 3 μM pepstatin (Sigma).
Sf9 cells expressing histidine-tagged TrkA498 796 (~7 litres of medium) were lysed by sonication in 100 ml of 50 mM NaKPO4 (pH 8.0), containing 300 mM NaCl, 5% (w/v) glycerol, 10 mM imidazole, 10 mM 2-mercaptoethanol, 0.5 mM PMSF and protease inhibitor cocktail (Roche).
Approximately 100 OD600 units of log-phase cells grown in thiamine-free EMM medium were lysed by glass bead beating in the lysis buffer (50 mM HEPES, pH 7.5, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 1 mM PMSF, 1 mM DTT, 0.05% NP-40, 1× Roche protease inhibitor cocktail).
For each of the two growth conditions (Wnt3a+ medium, Wnt3a− medium), cells were lysed by addition of TRIZOL reagent (Invitrogen, Carlsbad, CA) and total RNA isolated according to the manufacturer's protocol.
After removal of the medium, cells were lysed and cAMP accumulation was determined using the SMP004 kit from PerkinElmer according to the instructions of the manufacturers.
For Western blot analysis, 1-5×107 cells were stimulated with anti-CD40 beads (10 µl per 107 cells) in 0.4 ml culture medium; cells were lysed and beads were washed as above with 0.4 ml volumes of lysis buffer.
After an 8-h incubation period in complete or serum- and glucose-free medium, cells were lysed and deproteinized with an aqueous solution of 6% meta-phosphoric acid.
After an 8-h incubation in complete or serum- and glucose-free medium, cells were lysed with a modified RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC and 1% Triton X-100) supplemented with 1 mM NaF and with an antiprotease cocktail (Roche) containing 0.1 mM PMSF.
