Rabbit anti phospho nf κb p65

PBMCs from blood of healthy donors were seeded into 6-well plates at a density of 1 × 10⁶ cells/mL, pretreated with 2 μM JT11 for 1 h, in presence or not of CB2R antagonist SR144528 1 μM, and stimulated with 100 ng/mL LPS (Sigma-Aldrich) for 1 h. Subsequently, cells were harvested and lysed in RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 0.5% Triton X-100, 0.25% Nadeoxycholate, 0.1% SDS, 150 mM NaCl, 1mM EDTA and 5mM MgCl2, including proteases and phosphatases inhibitors). The total protein concentration in each sample was determined by Bradford assay. Protein lysates were analyzed in Western blot as previously described using rabbit anti-phospho-ERK1/2 (Cell Signaling Technology) and rabbit anti-phospho-NF-κB-p65 (Cell Signaling Technology). Immunoreactivity was detected using the ECL Western blotting detection system. As a control for loading of preparation, membranes were stripped and reprobed with rabbit anti-total ERK1/2 (Cell Signaling Technology) or mouse anti-β-tubulin mAbs (Sigma-Aldrich). In parallel experiments membranes were stripped and reprobed with rabbit anti-NF-κB-p65 or with anti-β-actin mAb (Sigma-Aldrich). Densitometric analysis was performed on Mac OS X (Apple Computer International), using NIH Image 1.62 software. The density of each band (absolute value) in the same gel was analyzed.

Transfected cells or grinded tumor tissues were lysed in modified RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% vol/vol NP-40, 1% n-Dodecyl β-D-maltoside, 0.25% wt/vol sodium deoxycholate, 1 mM DTT, and 1 × Roche complete Protease Inhibitor Cocktail) for 1 h at 4 ℃. The lysate was clarified by centrifugation for 20 min at 14,000 × g. The protein concentration was determined using a bicinchoninic acid assay and equal amounts of total protein from each of the samples was supplemented with 5 × SDS loading buffer, incubated at 95 ℃ for 5 min, subjected to SDS-PAGE, followed by western blot analysis. The following antibodies were used: rabbit anti-β-actin (Affinity; 1:5000), rabbit anti- AMPKα (Cell Signaling Technology; 1:1000), rabbit anti-Phospho-AMPKα (Cell Signaling Technology; 1:1000), mouse anti-E-cadherin (Cell Signaling Technology; 1:1000), rabbit anti-N-cadherin (Elabscience; 1:1000), rabbit anti-MMP3 (Proteintech; 1:1000), rabbit anti-GLI1 (Cell Signaling Technology; 1:1000), rabbit anti-GLI2 (NOVAS; 1:1000), goat anti-GLI3 (R&D; 1:1000), mouse anti-IκBα (Cell Signaling Technology; 1:1000), rabbit anti-NF-κB p65 (Cell Signaling Technology; 1:1000) and rabbit anti-Phospho-NF-κB p65 (Cell Signaling Technology; 1:1000).