3 3 dithiodipropionic acid di n hydroxysuccinimide ester

FTC-133 thyroid cancer cells were plated and let adhere 24 h before transfection with the vectors bearing the cDNA for wt, G129E or C124S PTEN. 36 h post-transfection the cells were treated as indicated, washed twice with ice-cold PBS, and harvested with Tris-Hcl buffer (50 mM Tris-HCl pH8), 1% NP-40, 150 mM NaCl) supplemented with phosphatases inhibitors (NaV, NaF) and proteases inhibitor cocktail (1μg/μl, Sigma). In each plate, 15 min before the ending of the treatment, 0.5 M of the chemical cross-linker 3-3’-dithiodipropionic acid di-(N-hydroxysuccinimide ester) (Sigma Aldrich) dissolved in DMSO was added. The same amount of protein (400-500 μg) was incubated with anti-HIS TAG antibody (2 μg) for at least 1 h at 4°C under rotation. In the case of the WRO thyroid cancer cells, which express endogenous wt PTEN, the cells were incubated with the cross-linker as above and the cell homogenate precipitated with anti-PTEN. To capture the immunocomplex, 50 μl of sepharose G beads (P3296, Sigma) were added to each sample and left under rotation overnight at 4°C. Immunocomplexes were then precipitated by centrifugation (1000 g) and eluted with 80 μl of Leammli buffer 1x at 95°C for 10 min. Equal volume of eluate was loaded on a SDS-containing polyacrylamide gels and immunoblotted with specific antibodies to reveal the presence of AKT and phosphoAKT in the immunoprecipitates.