Hypoxia inducible factor 1α hif 1α

The removed whole left lungs were used for western blotting, which was performed as described previously [17 (link), 18 (link)]. Polyvinylidene difluoride membranes were incubated with mouse monoclonal antibodies to β-actin (Santa Cruz) and heme oxygenase-1 (HO-1) (Novus Biologicals USA, Littleton, CO, USA) diluted to 1:1000 for 1 h at room temperature. Rabbit polyclonal antibodies to active caspase-3 (Cell Signaling Technology, Beverly, MA, USA), PPAR-γ (Cell Signaling Technology), voltage-dependent anion channel (VDAC) (Cell Signaling Technology), hypoxia-inducible factor-1α (HIF-1α) (Cell Signaling Technology), and apelin (Bioss Inc, Boston, MA, USA) were diluted to 1:500. Then, each membrane was incubated with a horseradish peroxidase-conjugated secondary antibody or IRDye® 680RD or IRDye®800CW (LI-COR, Inc., Lincoln, NE, USA) diluted to 1:10,000 for 45 min.

Following gene transfection, cells were collected and lysed in ice-cold RIPA extraction solution (Thermo, Rockford, IL, USA). Cell proteins were separated by 8–15% SDS-PAGE and transferred to PVDF membranes using a Mini Trans-Blot Cell and System (Bio-Rad, Hercules, CA). The membranes were blocked with nonfat milk and incubated overnight with primary antibodies diluted in TBST at 4°C. The following primary antibodies were used: FILIP1L, angiostatin, endostatin, and matrix metalloproteinases (MMP)-2 and -9 (Abcam, Cambridge, UK); phospho-Akt (Ser473), Akt, phospho-β-catenin (Ser33/37/Thr41), β-catenin, phospho-glycogen synthase kinase 3 beta (GSK-3β) (Ser9), GSK-3β, cleaved caspase-3, -7, -9, and hypoxia-inducible factor-1α (HIF-1α) (Cell Signaling, Danvers, MA, USA); and vascular endothelial growth factor (VEGF)-A and -D, and GAPDH (Santa Cruz Biotechnology). The membranes were developed using an ECL reagent (Amersham, Arlington Heights, IL, USA). Immunoblots were quantified using Multi-Gauge software (ver 3.0, Fujifilm, Tokyo, Japan).

Western blot was examined as reported [9 (link)]. The antibodies for IκBα (#4814), phospho-IκBα (#2859), IKKβ (#8943), phospho-IKKβ (#2078), p65 (#8242), phospho-p65 (#3033), phospho-PI3K (#4228), PI3K (#4249), phospho-Akt (#9611), Akt (#4691), hypoxia-inducible factor-1α (HIF-1α; #36169), cleaved-caspase3 (#9664), caspase3 (#9662) and GAPDH (#5174) were all bought from Cell Signaling Technology (Cell Signaling Technology Inc, Beverly, U.S.A.). The antibodies for Bcl-2 (#sc509) and Bax (#sc20067) were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, U.S.A.).