Anti phospho nf κb

Gut tissue samples were homogenized and lysed using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, Inc., St. Louis, MO, USA) supplemented with protease inhibitors. Protein concentrations were measured using the bicinchoninic acid (BCA) method. Equal amounts of proteins were loaded and separated on 10% SDS–PAGE, transferred to a nitrocellulose membrane and immunoblotted with indicated antibodies. Blots were visualized by enhanced chemiluminescence (ThermoFisher Scientific, Waltham, MA, USA). Antibodies used in this study were as follows: anti-Lcn2 (R&D System, Minneapolis, MN, USA), anti-NFκB (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-phospho-NFκB, and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA).

Western blot analysis was performed as described previously [33 (link)] with minor modifications. Briefly, aliquots of 30–60 µg of protein were subjected to 8–12% SDS-PAGE and transferred onto Hybond-P⁺ polyvinylidene difluoride membranes (Amersham, Buckinghamshire, UK). The membranes were incubated with the following primary antibodies: anti-ICAM-1 (ab225884, 1:1000, Abcam, Cambridge, UK), anti-VCAM-1 (ab106778, 1:1000, Abcam), anti-phospho-ERK (sc-7383, 1:1000, Santa Cruz Biotechnology), anti-total-ERK (sc-94 1:1000, Santa Cruz Biotechnology), anti-phospho-PKC (9375S, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-PKC (sc-10800, 1:1000, Santa Cruz Biotechnology), anti-phospho-PDK1 (3061S, 1:1000, Cell Signaling Technology), anti-PDK1 (3062S, 1:1000, Cell Signaling Technology), anti-phospho-STAT-3 (9131S, 1:1000, Cell Signaling Technology, anti-STAT-3 (4904S, 1:1000, Cell Signaling Technology), anti-HIF-1α (ab2185, 1:1000, Abcam), anti-phospho-NF-κB (8242S, 1:1000, Cell Signaling Technology), anti-NF-κB (8242S 1:1000, Cell Signaling Technology), and anti-β-actin (A2066, 1:2000, Sigma-Aldrich, St. Louis, MO, USA). The bound antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and an ECL western blotting detection reagent (Bio-Rad, Hercules, CA, USA).

The following antibodies (Abs) and reagents were used in present studies: anti-IL-1β (human specific, mouse specific, and human & mouse specific), anti-phospho NF-κB, anti-NF-κB, anti-phospho ERK, anti-ERK, anti-phospho p38, anti-p38, anti-phospho 4E-BP1, anti-4E-BP1, anti-phospho Mnk1, anti-Mnk1, anti-phospho eIF4E, anti-eIF4E, anti-phospho MK2, anti-MK2, and anti-streptavidin-HRP antibodies were purchased from cell signaling technology (Danvers, MA). Anti-β-actin, Dimethyl sulfoxide, Actinomycin D, Cyclohexamide, ultrapure E. coli O111:B4 LPS, Rapamycin, PD98059, and SB202190 were purchased from Sigma-Aldrich (St. Louis, MO). Anti-NLRP3 antibody was purchased from Adipogen (San Diego, CA). Torin 1 and FR180204 were purchased from Merck Millipore (Billerica, MA). CGP57380 and MK25 were purchased from TOCRIS (Ellisville, MO, USA) and Cayman Chemical (Ann Arbor, MI), respectively. Recombinant human M-CSF and mouse M-CSF were purchased from R&D system (Minneapolis, MN).