P akt

Mammary tumors were minced and resuspended in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Deoxycholate, 0.1% SDS and 50 mM Tris HCl, pH 8) with protease inhibitors, sonicated, spun down, and fat was removed before protein quantification. Immunoblots were performed using 20–60 μg of protein using the following antibodies, all from Cell Signaling Technology (Danvers, MA): HER2 (#4290T), p-HER2 (Y1248, #2247T), p-HER2 (Y1221/1222, #2243T), Akt (#4691T), p-Akt (S473, #4060T), p-Akt (T308, #13038T), actin (#3700T). The mouse phospho-RTK analysis was performed using the Proteome Profiler Mouse Phospho-RTK Array Kit (R&D Systems, #ARY014, Minneapolis, MN) according to the manufacturer’s instructions. Signal densities were quantified using NIH Image software and normalized as indicated in the figure legends and also, to the background.

Nuclear and cytoplasmic extracts from naive T cells cultured for 3 days were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce), as previously described^{21 (link)}. The purity of the nuclear and cytoplasmic fractions was verified by probing with antibodies against Lamin B1 (D4Q4Z; 1,000×; Cell Signaling Technology). Whole-cell extracts were immunoprecipitated with an anti-Raptor (24C12; 100×; Cell Signaling Technology), anti-Def6 (Rabbit polyclonal; 100×^{21 (link)}), anti-p62 (H-290; 50×; Santa Cruz), anti-TRAF6 (H274; 50×; Santa Cruz), or anti-HA (3F10; 50×; Roche Applied Science) antibodies. Antibodies to p-STAT3 (Y705; 1,000×), p-4E-BP (T37/46; 1,000×), 4E-BP (1,000×), p-S6K1 (S371; 1,000×), S6K1 (1,000×), p-AKT (S473; 1,000×), AKT (1,000×), p-AKT (T308; 1,000×), p-PRAS40 (T246; 1,000×), PRAS-40 (1,000×), p-AMPK (T172; 1,000×), AMPK (1,000×), p-Raptor (S792; 1,000×) and p62 (5114; 1,000×) were obtained from Cell Signaling Technology. Antibodies to IRF4 (M-17; 1,000×), TRAF6 (H274; 500×), and c-Myc (9E10; 500×) were obtained from Santa Cruz. Anti-Bcl6 antibody was obtained from BD (K112-91; 1,000×). Anti-Flag monoclonal antibody M2 (horseradish peroxidase (HRP)) was obtained from Sigma (1,000×).

Anti-Smad2 (#5339), p-Smad2 (Ser465/467 #3108), p-Akt (Thr308, #2965), p-Akt (Ser473, #4060), NFkB (#3037), p-Erk (Ser259 #9911), p-p38 (#4511), p-Jnk (#4668), p-Stat3 (#9145), p-Stat1 (#9171), Tgfbr2 (#3713), p-IRS1 (Tyr895, 3070), p-Smad1/5(#9511), p-Pten (Ser380/Thr382/383, #9549), Pten (D4.3 #9188), and Act-b (13E5, #4970) antibodies were purchased from Cell Signaling Technology. Anti-p-Akt (T308) antibody (# 658320) was purchased from R&D Systems. Anti-Mycn (B8.4.B, #sc-53993) antibody was purchased from Santa Cruz Biotechnology. Anti-pCKIIβ (S209) antibody (STJ90892) was purchased from St Joh’s laboratory. Anti-Gapdh antibody (10R-2932) was purchased from Fitzgerald Industries International. Western blot analysis was performed according to the standard procedure. Uncropped blots are included in Supplemetary Figs. 9–12.

Total protein extracts were obtained treating each tissue sample with total lysis buffer (Pierce Ripa buffer, Thermo Scientific, Rockford, IL, USA) supplemented with protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL, USA). After homogenization and centrifugation at 14,000 rpm for 15 min at 4 °C, the protein concentration was measured by a standard Bradford assay (Bio-Rad, Milan, Italy). Aliquots of 50 µg of total protein extracts from each sample were denaturated in 5× Laemmli sample buffer and loaded into 4–12% pre-cast polyacrylamide gels (Bio-Rad, Milan, Italy) for western blot analysis. Cannabinoid receptor I (Abcam, Cambridge, UK), cleaved caspase-3 (Asp175), Bax, ERK1/2, p-ERK1/2 (Thr202/Tyr204), p38α MAPK, p-p38 (Thr180/Tyr182) MAPK, Akt, p-Akt (Thr308), p-Akt (Ser473), β-actin (Cell Signaling Technology, Beverly, MA, USA) and Bcl2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), were used as primary antibodies. After overnight incubation, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Milan, Italy). The proteins were detected by chemiluminescence (ECL, Thermo Scientific, Rockford, IL, USA) and each protein-related signal was obtained using the Molecular Imager Chemidoc^TM (Bio-Rad, Milan, Italy) and normalized against β-actin protein expression.

Cells were plated in 6 well plates at a seeding density of 1,000,000 cells/well and treated with various concentration of C800 at different time points. Cells were harvested and lysed on ice for 5 min in lysis buffer (400 mM NaCl, 0.5% triton X-100, 50 mM tris pH 7.4) and sonicated for 40 s at 10 mA. Proteins were quantified and equal amount of proteins from each group were separated in 4–15% SDS-PAGE (BioRad (Sydney, NSW, Australia)) and transferred electrically onto PVDF membrane (BioRad (Sydney, NSW, Australia)). The membrane was blocked with 5% skim milk powder in TBST, washed thrice with TBST, and incubated with specific primary antibodies overnight at 4 °C. The membranes were blotted for cyclin D1, cyclin E1, CDK4, CDK6, CDK2, p21, p-Akt (Ser473), p-Akt (Thr308), p-mTOR (Ser2448), p-p44/42 MAPK (T202/T204), and p-p65 (Ser536) (Cell Signaling Technologies, Brisbane, QLD, Australia). Dilutions were made according to manufacturer’s protocol. Then, the membrane was washed thrice with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000 dilution, GE Technologies, Brisbane, QLD, Australia) for 1 h at room temperature. The proteins were detected using Clarity Western ECL detection kit (BioRad (Sydney, NSW, Australia)). α-Tubulin (monoclonal, 1:5000, clone B512, Sigma-Aldrich, St Louis, MO, USA) was used as a loading control.