Sepharose g beads

For immunoprecipitation, Ang2 (500 ng) and integrin αvβ5 (500 ng) were precleared with G-Sepharose beads (Millipore) at 4 °C for 1 h. Then, the complex and anti-integrin αvβ5 antibody (2 μg) were incubated with G-Sepharose beads at 4 °C overnight. The immune complexes were collected by centrifugation and washed with buffer three times. For immunoblotting, cells were collected and lysed in RIPA buffer with a protease inhibitor cocktail. Protein lysates were resolved by SDS polyacrylamide gel and transferred onto nitrocellulose membrane. The membranes were incubated with primary antibodies (1 : 1000) overnight at 4 °C and secondary antibodies (1 : 10 000) for 1 h at room temperature. The membranes were incubated with enhanced chemiluminescent substrate (Pierce) and exposed to a film or LAS 4000 (GE Healthcare, Piscataway, NJ, USA).

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.

DR-Wildtype (ATCC CRL2977) and GCN2-KO-DR (ATCC CRL2978) MEFs, purchased from American Type Culture Collection (ATCC), were maintained in DMEM containing 10% (vol/vol) FBS (Invitrogen), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM L-Glutamine, 1 mM sodium pyruvate, and 0.05 mM 2-mercaptomethanol. Sodium thioglycolate (Sigma-Aldrich) was used to generate thioglycolate-elicited peritoneal macrophages. LPS (Sigma-Aldrich) and ATP (Sigma-Aldrich) were used at a concentration of 500ng/ml and 5mM, respectively, to stimulate BMDMs, peritoneal macrophages, or J774A.1 cells cultured in DMEM containing 10% (vol/vol) FBS (Invitrogen), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM L-Glutamine, and 1 mM sodium pyruvate. HF hydrobromide (trans-[±]-7-Bromo-6-chloro-3-[3-(3-hydroxy-2-piperidinyl)-2-oxopropyl]-4[3H])quinazolinonemonohydrobromide (Sigma-Aldrich); 3-MA (Sigma-Aldrich), and Wortmannin (Sigma-Aldrich) were used at a concentration of 5 mM or 5 μM. Cycloheximide and Act-D (Sigma-Aldrich) were used at 1 μg/ml and 10 μg/ml. Rapamycin (Sigma-Aldrich) was used at a concentration of 200 nM. Lipofectamine 3000 was from Life Technologies (L3000015) and Sepharose G beads from Sigma-Aldrich. pCMV6 IL-1β was purchased from Origene.