Antibody against p65

Human recombinant TNFα protein was purchased from PeproTech (Rocky Hill, NJ, USA). Src-inhibitor saracatinib, JAK-inhibitor tofacitinib, mitogen-activated protein kinase-inhibitor SB203580, STAT3-inhibitor S3I-201, p65-inhibitor JSH-23, IKK-inhibitors TPCA-1 and IKK-16 were perchased from Selleckchem (Houston, TX, USA), JAK-inhibitor PDTC was perchased from Abcam (Cambridge, MA, USA). Antibody against HK2, phospho-IKKα/β (Ser176/180) and phospho-IKKε (Ser172) were purchased from Cell Signaling Techonology (Beverly, MA, USA), antibody against YAP was purchased from NOVUS Biologicals (Littleton, CO, USA), antibody against CD68 was pursed from Abcam, antibody against p65 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against β-actin, GFP, Myc, Flag, HA and TEAD4 were purchased from Sigma-Aldrich. Antibody against TNFR1, IKKα, IKKβ and IKKε were purchased from Abclonal Technology (Wuhan, China). Antibody against pan-phosphorylated serine/threonine was purchased from BD Biosciences (San Jose, CA, USA).

To assess the NF-κB nuclear translocation, the THP1-derived macrophages and HAoECs grown in a 4-well Slide&Chamber (Watson Bio Lab, Tokyo, Japan) for 2–3 days were washed twice with PBS and incubated with GIP_HUMAN[22–51] (10^–7 M) for 60 min. The cells were fixed with 4% paraformaldehyde for 15 min, blocked with Blocking One (Nacalai Tesque Inc., Kyoto, Japan), incubated for 60 min with an antibody against p65 (1:500, Santa Cruz Biotechnology, Inc., CA, USA) at room temperature and incubated for 30 min with the biotinylated goat anti-rabbit antibody (1:3000, Vector Laboratories Inc., Burlingame, CA, USA). The avidin–biotin-peroxidase complex was formed by using the Vectastain ABC Kit (Vector Laboratories Inc.) and visualized by using the DAB peroxidase substrate kit (Vector Laboratories Inc.).