Anti hif 1 alpha antibody

Hyaluronic acid (HA) (MW: 575 kDa) was received as gift from Hanmi Pharmaceutical Co. (Pyeongtaek, Korea), 1,4-butanediol diglycidyl ether (BDDGE, MW: 202 Da), α-MEM and sodium butyrate (NaB) were purchased from Sigma-Aldrich (St. Luis, MO, USA). Sodium hydroxide (MW: 40 Da) was purchased from Yakuri Pure Chemical Co. (Kyoto, Japan). Poly(dimethyl siloxane) (PDMS) (184 Sylgard) was purchased from Dow Corning (Auburn, MI, USA). Penicillin-streptomycin was purchased Lonza Korea (Basel, Switzerland). Cell counting kit-8 (CCK-8) solution was bought from Dojindo Laboratories (Kumamoto, Japan). Live & dead viability/cytotoxicity kit for mammalian cells was purchased from Invitogen (Carlsbad, CA, USA). Dimethyloxalylglycine (DMOG) was procured from Cyman Chemical (Ann Arbor, MI, USA). Anti-RUNX2 antibody, anti-HIF-1-alpha antibody, anti-beta actin antibody, anti-osteocalcin antibody and Goat Anti- Mouse IgG H&L (HRP) were bought from Abcam (Cambridge, UK). RIPA buffer, protease inhibitor, and phosphatase inhibitor were purchased from Sigma-Aldrich (USA).

L-Nitro-arginine-methyl-ester (L-NAME) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany; batch no. N5751-10), and sodium pentobarbital was purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Anti-HIF-1 alpha antibody, anti-PTEN antibody, anti-VEGFA antibody, anti-PI3K p85 antibody, anti-PI3K p85 (phospho Y607) antibody, and anti-AKT1 (phospho S473) antibody were purchased from Abcam (batch no. Ab1; ab32199; ab1316; ab86714; ab182651; ab81283; dilution 1 : 200; 1 : 100; 1 : 100; 1 : 100; 1 : 200; 1 : 200); TRIB3 polyclonal antibody, AKT (L321) polyclonal antibody, and AKT (phospho-T308) polyclonal antibody were purchased from Bioworld (batch no. BS60451; BS1502; BS4647; dilution 1 : 100; 1 : 100; 1 : 100).

After completion of oxygen measurements as described in Section 2.3, the devices were opened, and the medium was aspirated. The cells were immediately washed with PBS buffer and fixed with 4% formaldehyde ( Sigma-Aldrich, Burlington, MA, USA, cat. 252549) in PBS for 15 min at room temperature. After further washing, cells were permeabilized with 0.1% solution of Triton X-100 ( Sigma-Aldrich, Burlington, MA, USA, cat. X-100) in PBS for 5 min to allow for staining of intracellular proteins, and then washed again. All washes were performed five times using PBS. Cells were blocked against unspecific binding with 5% Bovine Serum Albumin ( Sigma-Aldrich, Burlington, MA, USA, cat. A7906) in PBS for 1 h. Subsequently, cells were stained overnight at 4 °C with an anti-HIF-1 alpha antibody (Abcam, cat. ab179483) at a 1:500 dilution in the blocking buffer. After washing, secondary antibody (anti-rabbit-Alexa 488 IgG, Thermo Fisher Scientific, Waltham, MA, USA, cat. A21206) at a 1:1000 dilution was added for 1 h, at room temperature. Finally, cells’ nuclei were stained with DAPI ( Sigma-Aldrich, Burlington, MA, USA, cat. D9542) for 15 min and then the cells were washed again. Imaging was performed with Leica SP5 confocal microscope (Leica Camera, Wetzlar, Germany) with Leica LAS AF software, using HC PL APO 20x/0.70 objective. At least five fields of view were captured per culture chamber.