Phospho tie2

Cells were washed with PBS then lysed using radioimmunoprecipitation assay (RIPA) buffer (Boston Bioproducts) with protease inhibitor and phosphatase inhibitor cocktail (Roche). The protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientifc). Cell lysates were resolved by SDS-PAGE, transferred to a PVDF membrane (Immobilon-P, Millipore), and then blocked in 5% nonfat dried milk for 1 hour. The membrane was then analyzed by immunoblotting with antibodies against the following: phospho-c-ABL (Y245) (0.4 μg/ml; Cell Signaling), c-ABL (1.5 μg/ml; Cell Signaling), phospho-TIE2 (0.002 μg/ml; Cell Signaling), TIE2 (1 μg/ml; Abcam), phospho-AKT (Ser473) (0.05 μg/ml; Cell Signaling), AKT (0.04 ug/mL; Cell Signaling), Podocalyxin (1 ug/mL; R&D Systems), ICAM2 (0.056 μg/ml; Cell Signaling) and β-Actin (1 μg/ml; Sigma). Membranes were incubated with peroxidase-conjugated secondary antibodies (1:5000 Vector Laboratories). Antigen-antibody complexes were visualized using Immobilon Forte Western HRP Substrate (Millipore) on a ChemiDoc™ Gel Imaging System (Biorad) or chemiluminescent sensitive film.

Keratinocytes with required confluence were pre-incubated with 0.1–20 µg/mL lupeol for 24 h. After 24 h incubation, cells were then lysed in ice-cold lysis buffer supplemented with protease inhibitors and phosphate inhibitors and centrifuged at 12,000× g for 5 min. An aliquot of cell lysate was separated by 10% SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred to a polyvinylidene difluoride membrane. After blocking in a 5% skim milk powder solution, the membranes were incubated with B-actin, phosphorylated (P) forms of p38 (Tyr182) and p38 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) phospho-Akt (Ser473), Akt, phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2), Tie2 (D9D10), phospho-Tie2 (Tyr992), NF-κB-p65 (Ser536), MMP-2 (Cell Signaling Technology, Beverly, MA, USA), and Keratin 16 (Thermo Fisher Scientific, Waltham, MA, USA) overnight at 4 °C. The next day, all membranes were washed and incubated in secondary antibody for 1 h at room temperature. Proteins were detected by the ECL (electrochemiluminescence) detection system, (Amersham Biosciences, Little Chalfont, UK) and analyzed by ImageJ software.