Phospho pak1

Proteins in cell lysates were detected with antibodies against phospho-PAK1 (Santa Cruz Biotechnology), PAK1, phospho-AKT, AKT, HIF1α (BD Biosciences, North Ryde, Australia), and GAPDH. Antibodies were from Cell Signalling Technology (Arundel, Australia), unless otherwise stated. Bound antibodies were visualized using ECL reagents (GE Healthcare, Amersham, UK), and the density of each band was analysed using Multigauge computer software (Berthold, Bundoora, Australia). HIF1α expression was determined in cells cultured under normoxia or hypoxia (1 % O₂).

Punicalagin (purity >98%) was obtained from Cayman Chemical (Ann Arbor, United States) and dissolved in dimethyl sulfoxide (DMSO). Human recombinant tumor necrosis factor alpha (TNF-α), VEGF165, and ELISA kits for interleukin-6 (IL-6), interleukin-8 (IL-8), ICAM-1, VCAM-1 and monocyte chemotactic protein 1 (MCP-1) were obtained from Cell Signaling Technology (Danvers, MA, United States). Collagenase II, DMEM and Penicillin-Streptomycin Mixed Solution were purchased from Thermo Fisher Scientific (Gibco, Grand Island, United States). Antibiotics, trypsin/EDTA, and phosphate buffered solution (PBS) were purchased from Solarbio (Beijing, China). EBM-2 and EGM-2 media were obtained from LONZA (United States). Antibodies against VCAM1, ICAM1, IKBα, phospho-IKBα, phospho-VEGFR2, VEGFR2, phospho-PAK1, PAK1 and NF-κBp65 were obtained from Santa Cruz Biotechnology. Sterile plastic material was obtained from Corning Inc. (NY, United States). Fetal bovine serum (FBS), reagents and materials for Western Blot analysis were purchased from Sigma-Aldrich (St. Louis, MO, United States).