Anti interleukin 6 il 6

Protein lysates were extracted according to a previously described method (Hwang et al. 2018 ; Kim et al. 2018 (link)). Dissected hippocampal tissues were homogenized in 400 μl per 1 g concentration of RIPA buffer (Cell Signaling Technology, Beverly, MA, USA) with 1 mM PMSF (Sigma-Aldrich Inc.) on ice. The homogenized sample was incubated on ice for 20 min. Incubated sample was centrifuged at 14,000 g for 10 min at 4°C, and supernatants were collected. Protein content was measured using a micro-drop plate reader (Thermo Fisher Scientific). NF-кB and IкBα in the hippocampus were detected using nuclear/cytosol fractionation kit (BioVision Inc, Milpitas, CA, USA) according to the manufacturer’s instructions. The following primary antibodies (1:1000 dilution) were selected to react overnight at 4°C: mouse anti-β-actin, anti-TNF-α, anti-interleukin-6 (IL-6), anti-proBDNF, anti-BDNF, anti-Iba-1, rabbit anti-TrkB, anti-NF-κB, and anti-IκBα (Santa Cruz Biotechnology). Subsequently, membranes were incubated for 1 h with attempt secondary antibodies (1:2000; Vector Laboratories). Blot membrane was detected using the HRP-conjugated IgG (Vector Laboratories) and the enhanced chemiluminescence detection kit (Bio-Rad, Hercules, CA, USA). Detected bands were quantified by Image-Pro® plus image analysis system (ver. 6.0, Media Cybernetics Inc., Silver Spring, MD, USA).

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.