Phospho nf κb p65 ser536

Antibodies used were phospho-IkBa (Ser32/36) (9246, Cell Signaling), phospho-SAPK/JNK (Thr183/Tyr185) (9251, Cell Signaling), SAPK/JNK (9252, Cell Signaling), IkBa (SC-371, Santa Cruz), phospho-IKKα/β (Ser176/180) (2697, Cell Signaling), Phospho-p38 MAPK (Thr180/Tyr182) (9215, Cell Signaling), Phospho-NF-κB p65 (Ser536) (3033, Cell Signaling), tubulin (ab7291, Abcam), actin (AAN01-A, Cytoskeleton), BiP (610978, BD Biosciences), CHOP (MA1-250, ThermoFisher), SLC39A7 (19429-1-AP, Proteintech), TNFR1 (sc-8436, Santa Cruz), TRAIL-R1/DR4 (42533, Cell Signaling), RIPK1 (610458, BD Bioscience), RIPK3 (12107, Cell Signaling), GFP (sc-69779, Santa Cruz), FADD (610399, BD Biosciences), phospho-MLKL (Ser385) (ab187091, Abcam), phospho-RIPK1 (S166) (65746, Cell Signaling), phospho-RIPK3 (S227) (ab209384, abcam), cleaved Caspase-3 (Asp175) (9661, Cell Signaling), V5 (R960-25, Invitrogen or ab9116, abcam), PDIA2 (ab2792, abcam), TNIP1/ABIN-1 (4664, Cell Signaling), cIAP1/BIRC2 (7065, Cell Signaling), cIAP2/BIRC3 (3130, Cell Signaling) and LAMP1 (ab25630, abcam). The secondary antibodies used were goat anti-mouse HRP (115-035-003, Jackson ImmunoResearch), goat anti-rabbit HRP (111-035-003, Jackson ImmunoResearch), Alexa Fluor 680 goat anti-mouse (A-21057, Molecular probes) and IRDye 800 donkey anti-rabbit (611-732-127, Rockland).

Anti-caspase-8 monoclonal antibody C15 recognizes the p18 subunit of caspase-8. Anti-FLIP monoclonal antibody NF6 recognizes the N-terminal part of c-FLIP. Anti-FADD monoclonal antibody 1C4 recognizes the C-terminal part of FADD46 (link). Antibodies against Caspase-3, caspase-9, p65, Phospho-NFκB p65 (Ser536), cIAP1, cIAP2, XIAP, Bcl_XL, and Smac were obtained from Cell Signaling. Antibodies specific for Bcl-2 were purchased from Santa Cruz Biotechnology, RIPK1 from BD Bioscience, Trx and Tom20 from Abcam, β-Actin from Genetech and Tubulin from Sigma.

LPS (Escherichia coli O111:B4) and 5-HMF were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Gibco (ThermoFisher, Gaisburg, MD, USA). Fetal bovine serum (FBS) was purchased from Lonsera (Lonsa Science SRL, Guichon, Uruguay). MTT cell proliferation and cytotoxicity assay kit was purchased from Phygene life sciences (Fuzhou, China). The primary rabbit monoclonal antibodies against p38 MAPK (D13E1) XP^®, SAPK/JNK Antibody, p44/42 MAPK (ERK1/2) (137F5), phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP^®, phospho-SAPK/JNK (Thr183/Tyr185) (81E11), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E) XP^®, Akt (pan) (C67E7), mTOR (7C10), phospho-Akt(Ser473) (D9E) XP^®, phospho-mTOR (Ser2448) (D9C2) XP^®, IκBα (44D4), NF-κB p65 (D14E12) XP^®, phospho-IκBα (Ser32) (14D4) and phospho-NF-κB p65 (Ser536) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Myricetin (≥98%) purchased from Herbpurify (Chengdu, China) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) under sterile conditions to make a stocking solution of 40 mg/ml. The T-PER Tissue Protein Extraction Reagents and Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA) were used for total protein extraction and the measurement of total protein concentration, respectively. The antibodies used in this study were as follows: polyclonal rabbit anti-ERK1/2 (Proteintech), phospho-ERK1/2 (Thr202/Thr204) (Arigo), JNK (Proteintech), phospho-SAPK/JNK (Thr183 (221)+Thr185 223) (Arigo), p38 MAPK (Cell Signaling Technology), phospho-p38MAPK (Thr180/Tyr182) (Cell Signaling Technology), NF-κB p65 (Cell Signaling Technology), phospho-NF-κB p65 (Ser536) (Cell Signaling Technology), IKK alpha, phospho-IKK alpha (Thr23) (Arigo), and monoclonal mouse anti β-actin (Proteintech) for inflammation reaction analysis and Staphylococcal α-toxin (Sigma-Aldrich) and HRP-conjugated secondary antibodies (Proteintech) for Hla production analysis. All of these antibodies were used as recommended by the manufacturers.

Cells were grown to 70–80% confluency in RPMI-1640 supplemented with 10% FBS in 4-chamber polystyrene vessel tissue culture-treated glass slides and then treated with 5-FU as indicated in each experiment. After the cells were exposed to 50 µM of 5-FU for 4 h to see an early transcriptional response, they were fixed in 4% paraformaldehyde, permeabilized using 0.2% Triton X-100 in PBS, and stained with DAPI (0.6 µM DAPI, 50 µl RNase, and 5 ml PBS) at room temperature for 12 min. Cells were then incubated with the following primary antibodies: anti-NF-κB p65, phospho-NF-κB p65 (Ser536), and phospho-p53 (Ser15) (Cell Signaling Technology Japan, Tokyo, Japan), and p53 (Thermo Scientific, Kalamazoo, MI, USA). Finally, the cells were incubated with either Alexa Fluor488- or 568-conjugated secondary antibody (Life Technologies Japan, Tokyo, Japan). A BX43 fluorescent microscope (Olympus, Tokyo, Japan) was used for image acquisition.

Proteins for western blot analysis were extracted from the renal cortex by lysing with NETN150 (0.5% NP-40, Tris PH 8.0 50 mM, NaCl 150 mM). The proteins were quantified by Bradford, and the samples were separated by SDS PAGE. Gels were transferred to the nitrocellulose membrane (Axygen, Union City, CA) and then blocked with 5% nonfat milk for 1 hour at room temperature, the membrane was incubated with primary antibodies overnight at 4°C, followed by horseradish peroxidase- (HRP-) linked secondary antibody. The antibodies against TGF-β1 (ab169771) and NF-κBp65 (ab7970) were purchased from Abcam, and phospho-NF-κBp65 (Ser536) was bought from Cell Signaling. After usage of Immobilon Western Chemiluminescent HRP Substrate kit (Millipore Corporation, Billerica, MA), the band was quantified with ImageJ (National Institutes of Health, Bethesda, MD, USA).

Cells were collected in lysis buffer containing protease and phosphatase inhibitors for protein isolation. We prepared cellular extracts by sonication. And total protein concentrations were determined for the Western blot analyses. Proteins were separated on 4%-20% Tris-Glycine gels (Invitrogen, Carlsbad, CA, USA) and transferred to nitrocellulose membranes. After the membranes were blocked in TBST (10 mM Tris-HCl buffer, pH 8.0; 150 mM NaCl; and 0.1% Tween 20) and 5% (w/v) BSA at room temperature for 60 mins, they were incubated overnight at 4 °C with antigen-specific primary antibodies. The primary antibodies used were against phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, Danvers, MA, USA). The blots were under incubation with species-specific HRP-conjugated secondary antibodies for 2 hours at room temperature. And then, the proteins were visualized through incubation with a chemiluminescent substrate kit (Thermo Fisher Scientific, Waltham, MA, USA).