Investigating Anti-inflammatory Signaling Pathways

Monoclonal antibodies (mAb) against HO-1 and phospho-p65^Ser536 were from Abcam (Cambridge, UK). E-selectin mAb 1.2B6, ICAM-1 mAb 6.5B5 and VCAM-1 mAb 1.4C3 were generated in house. CD31 mAb P2B1 was generated in house from a clone purchased from Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA. The following antibodies were purchased from Cell Signaling (Danvers, MA) AMPKα, phospho-AMPKα^Thr172, CREB, phospho-CREB^Ser133. Antibodies against p65 and Nrf2 were from Santa Cruz Biotechnology (Heidelberg, Germany), α-tubulin Sigma-Aldrich (Poole, UK) and GAPDH Millipore (Watford, UK). 2,5-Dimethyl-Celecoxib was synthesized as described^{58 (link)}. Celecoxib was purchased from Toronto Research Chemicals (Ontario, Canada), naproxen and ibuprofen from Tocris Bioscience (Bristol, UK). The concentrations of all three drugs used in vitro (Celecoxib 1–10 µM, naproxen and ibuprofen 1–100 μM) included concentrations that can be achieved therapeutically in humans^{30 (link),38 (link),59 (link),60 (link)}. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) and Compound C were from Millipore, hemin and sulforaphane from Sigma-Aldrich, TNFα and IL-1β from R and D Systems (Abingdon, UK).