Transient transfections of β-catenin, β-galactosidase and Wnt reporter plasmids were performed using the Fugene reagent 24 hours after initial plating (Roche Molecular Biochemicals).

Separated proteins were electroblotted to PVDF membranes according to manufacturer's recommendations (Roche Molecular Biochemicals).

A 1:2000 dilution of anti-Elk-1 Ab (Santa Cruz Biotechnology) and a 1:5000 dilution of goat anti-rabbit secondary Ab (Roche Molecular Biochemicals) were used.

Limited proteolysis of Ppt1p Recombinant protein was incubated with either trypsin (Roche Molecular Biochemicals) or subtilisin (Roche Molecular Biochemicals) in the absence or presence of 250 μM lipid for 0 to 30 min at 30 C, as previously described [ 19 ] .

Following extensive washing in TBST, bands were visualized by the enhanced chemiluminescence method (Roche Molecular Biochemicals) and quantitated by laser densitometry.

In addition, after infection with Ad-GFP or Ad-GFP/Rz-IGF2R and treatment with TNF, cell viability was assessed using the MTT assay Kit (Roche Molecular Biochemicals) and cell apoptosis was determined using the Cell Death Detection ELISA Kit assay (Roche Molecular Biochemicals) according to the manufacturer's protocol.

The expression of HA-Ppt1p was detected by immunoblot using the 12CA5 monoclonal anti-HA antibody (Roche Molecular Biochemicals).

Apoptosis assay DT40 cells were treated for 24 h and flow cytometry analyses of apoptotic cells was performed using the TUNEL in situ cell death detection kit (Roche Molecular Biochemicals).