Anti phd2

HOK cells and mucosal epithelia were denatured in laemmli buffer, centrifuged and then heated for 10 min at 95 °C. Proteins were separated by SDS-PAGE, and then electroblotted onto PVDF membranes (Millipore). Western blot analyses were carried out as described [32 (link)]. The following primary antibodies (1:1000 dilution) were used in this study: anti-IKKβ, anti-phospho-NF-κB p65, anti-NF-κB p65 from Cell Signaling, anti-Toll-like receptor 4 (TLR4), anti-IFNγ, anti-IL-1β, anti-TNFα, anti-interleukin 6 (IL-6), anti-VDR and anti-β-actin from Santa Cruz Biotechnology, anti-HIF-1α from Millipore, anti-PHD1, anti PHD2, and anti-VHL from Abcam.

Radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China) was used to extract total protein from BMSCs. Quantification of proteins samples was performed by bicinchoninic acid (BCA) method (Solarbio, Beijing, China). After denaturation, the protein samples were then subjected to electrophoresis and transferred on a polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After electrotransfer, skim milk was used to block the membrane, followed by addition of primary antibodies (anti-HIF-1α and anti-PHD2, Abcam) for overnight incubation. Then, the secondary antibodies were used to probe the proteins. Bands were exposed by the enhanced chemiluminescence (ECL) method (General Electric, Boston, MA, USA) and analysis of bands’ gray value was achieved by Image J Software.