Anti hif 1α rabbit polyclonal antibody

LPS derived from Escherichia coli strain 055: B5, and the NF-κB inhibitor BAY 11–7028 were purchased from Sigma-Aldrich. Recombinant human TNF-α was obtained from PeproTech. Poly (I:C) was obtained from InvivoGen. The following antibodies were used for western blotting: an anti-TLR4 rabbit polyclonal antibody (Abcam); anti-TLR3 rabbit polyclonal antibody (Cell Signaling Technology); anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (Bioworld Technology); anti-HIF-1α rabbit polyclonal antibody (Cell Signaling Technology), anti-VEGF rabbit polyclonal antibody (Abcam). Protein standards were obtained from SunShineBio. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) was purchased from Immunology Consultants Laboratory (Newberg, OR, USA). An anti-p65 rabbit polyclonal antibody (Cell Signaling Technology) and goat anti-rabbit IgG (Bioworld Technology) were used for immunofluorescence.

Protein extraction was performed using lysis buffer (10% glycerol, 10 mM Tris-HCl (pH7.5), 1 mM EDTA, 400 mM NaCl, 0.5% NP40, 4 μg/mL aprotinin, PMSF, proteasome inhibitor MG-132, and 1mM DTT). Total protein (10 µg) was electrophoresed on a 10% polyacrylamide gel and then electroblotted at 300 mA for 90 min on a nitrocellulose membrane (Invitrogen, Waltham, MA, USA). Western blotting was used to confirm the expression of HIF-1α and β-actin proteins, which were detected using anti-HIF-1α rabbit polyclonal antibody (1:1000) (Cell Signaling Technology, #3716, Danvers, MA, USA) and β-actin mouse monoclonal antibodies (1:1000) (A5316; 1:1000; Sigma, St. Louis, MO, USA), respectively. β-actin was used as a loading control. The signals were detected using the ECL Select Western Blotting Detection System (GE Healthcare Life Sciences, Chicago, IL, USA) and Image Quant LAS 4000 (GE Healthcare Life Sciences).