P nf κb

Total proteins were extracted using 100 μl lysis buffer form cells. After measurement of the quality and concentrations of the proteins, 25 μg samples were loaded and separated in 10% sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes through electroblotting. Then membranes were blocked with 5% skim milk for 1 h at room temperature, followed by incubation with P-gp (Cat. No. MA5-13854, Invitrogen, Carlsbad, CA, USA), MRP1 (SC-18835), Survivin (sc-17779), MMP2 (sc-13594), pSer473-Akt (sc-293125), Akt1 (sc-5298), p-ERK (sc-7383), ERK (sc-514302), pSer33/37-β-catenin (sc-57535), β-catenin (sc-7963), p-NFκB (sc-271908), NFκB (sc-8414), p-mTOR (sc-293089), mTOR (sc-517464), and GAPDH (SC-47724) antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4 °C in a dilution of 1:1000. Membranes were washed three times with phosphate-buffered saline Tween-20 and incubated with HRP-conjugated secondary antibody (Merck & Co., Inc., Kenilworth, NJ, USA) for another 1 h in a dilution of 1:2500. Immunoreactivity was detected using the Western Lighting Ultra (Thermo Fisher Scientific, Waltham, MA, USA).

Mouse monoclonal antibodies (STAT3, STAT5, ERK1/2, Akt, NF-κB, IL-6, actin, p-ERK1/2, and p-p38), rabbit polyclonal antibodies (cleaved IL-1β, TLR-2, TLR-4, TLR-5, p38, p-STAT3, p-STAT5, p-Akt1, and p-NF-κB), and goat polyclonal antibodies (TNF-α and IL-1β) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The samples derived from cells and lung homogenates were lysed in radioimmunoprecipitation assay (RIPA) buffer, separated by electrophoresis on SDS-PAGE gels, and transferred to nitrocellulose transfer membranes (GE Amersham Biosciences, Pittsburgh, PA). Proteins were detected by Western blotting using primary Abs at a concentration of 1/200 and were incubated overnight (51 (link)). Labeling of the first Abs was performed using relevant secondary Abs conjugated to horseradish peroxidase (HRP) (Santa Cruz Biotechnology), which were detected using ECL regents (Santa Cruz Biotechnology) and quantified using Quantity One software (Bio-Rad).

Urolithin A (≥97% purity by HPLC, Figure 1A) was purchased from Sigma-Aldrich (St. Louis, MO, USA), cell viability assays (MTT), and poly(I:C) were purchased from InvivoGen (San Diego, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN, USA). DAPI solution was purchased from Sigma-Aldrich (St. Louis, MO, USA). The inhibitor PD98059 was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Antibodies against β-actin, TLR3, TRIF, IRF, STAT1, NF-κB, I-κB, COX-2, iNOS, Nrf2, and phosphorylated IRF (pIRF), STAT1, NF-κB (pNF-κB), and IκB (pIκB) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against JNK, ERK, p38, and phosphorylated JNK (pJNK), ERK (pERK), and p38 (pp38) were purchased from Millipore (Billerica, MA, USA).

Jejunae from experimental animals was fixed in 10% formalin, processed and embedded. Immunohistochemistry for phospho-relA was performed on tissues as previously described [33 (link)]. Tissues were sectioned at 4 μm on a graded slide, deparaffinized in xylene, rehydrated in graded ethanols, and rinsed in Tris-phosphate-buffered saline (TBS). Heat-induced antigen retrieval was performed in a microwave at 98°C in 0.01 M citrate buffer. Primary antibody (p- NF-κB Santa Cruz Biotechnology (#sc-101749) 1:400) was exposed overnight followed by secondary anti-rabbit antibody (DAKO (#K4003)). Immunoreactivity was visualized by incubation with chromogen diaminobenzidine (DAB) for 5 minutes. Tissue sections were counterstained with hematoxylin, dehydrated through graded ethanols and xylene, and cover-slipped.

The MSCs were lysed using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) to obtain total cellular protein. Cell lysates (20 µg of total protein) were separated on 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred to the nitrocellulose membrane. After the blots had been washed with TBST (10 mM Tris-HCl (pH 7.60), 150 mM NaCl, 0.05% Tween-20), they were blocked with 5% skimmed milk for 1 h and incubated with appropriate primary antibodies at the dilutions recommended by the supplier. Antibodies against GRP78, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, IRE1α, CHOP, p-IRE1α, NF-κB, p-NF-κB, JNK, p-JNK, p38, p-p38, BCL-2, BAX, cleaved caspase-3, cleaved PARP-1, PrP^C, MnSOD, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The membranes were then washed, and primary antibodies were detected using goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).