Irak4

HEK293 cells (ATCC number CRL-1573) and RAW264.7 cells (ATCC number TIB-71) were acquired from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Dimethyl sulfoxide (DMSO), carboxymethylcellulose (CMC), 3-(4,5-dimethylthiazol,2-yl)-2,5-diphenyltetrazolium bromide (MTT), polyethylenimine (PEI), sodium dodecyl sulfate (SDS), ranitidine, dexamethasone (dexa), L-NAME, kaempferol, genistin, apigenin, lipopolysaccharide (LPS, E. coli 0111:B4), pam3csk4, and poly (I:C) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). RPMI 1640, DMEM, trypsin, PBS, and penicillin-streptomycin were received from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) was acquired from Biotechnics Research, Inc. (Irvine, CA, USA). TRIzol reagent was purchased from MRCgene (Cincinnati, OH, USA). Primers used for semiquantitative RT-PCR and qPCR were purchased from Macrogen Inc. (Seoul, Korea). Phospho-specific or total-protein antibodies against p65, p50, IκBα, PI3K/p85, Src, c-Fos, c-Jun, JNK, ERK, p38, MKK4, MKK7, MEK1/2, TAK1, IRAK1, IRAK4, hemagglutinin (HA), and β-actin were acquired from Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Constructs for signaling proteins HA-Src and HA-TAK1 were used as previously reported [24 (link),26 (link)].

Anthraquinone-2-carboxylic acid (AQCA, Figure 1(a)), indomethacin (Indo), ranitidine (RT), arachidonic acid, sodium carboxyl methylcellulose (Na CMC), phorbol-12-myristate (PMA), acetylsalicylic acid (ASA), and lipopolysaccharide (LPS, E. coli 0111:B4) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). SB203580 (SB), PP2, and piceatannol (Picea) were obtained from Calbiochem (La Jolla, CA, USA). All other chemicals used in this study were of analytical grade from Sigma Chemical Co. Phosphospecific or total antibodies that were raised against Src (Cat. numbers 2101 and 2102), spleen tyrosine kinase (Syk) (Cat. numbers 2711 and 2712), p38 (Cat. numbers 4631 and 9212), c-Jun N-terminal kinase (JNK) (Cat. numbers 9251 and 9252), cyclooxygenase- (COX-) 2 (Cat. number 4842), interleukin-1 receptor-associated kinase 4 (IRAK4) (Cat. number 4363), and β-actin (Cat. number 4967) were obtained from Cell Signaling (Beverly, MA, USA). RAW264.7 and HEK293 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Myeloperoxidase (MPO) activity colorimetric assay kit was obtained from Biovision (Milpitas, CA, USA). Luciferase constructs that contained the binding promoters for NF-κB and AP-1 were gifts from Professor Hae Young Chung (Pusan National University, Pusan, Korea) and Addgene (Cambridge, MA, USA).

Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Gibco-BRL (USA). Fetal bovine serum (FBS) was from Hyclone Lab Inc. (USA). LPS (Escherichia coli 0111:B4), sulforhodamine B (SRB), prodium iodide (PI) and CAPE were from Sigma Co. (USA). Primary antibodies against TLR4, NF-κB p65, β-actin and secondary antibody (horseradish peroxidase) were from Santa Cruz Biotechnology (USA). Primary antibodies against MyD88, TRIF, IRAK4, LC3B, PARP and procaspase 3 were purchased from Cell Signaling Technology (USA). Secondary antibody for immunofluorescence, donkey anti-rabbit IgG Alexa Fluor-488 was purchased from Life Technologies (USA). Nitric oxide (NO) kit was from Nanjing Jiancheng Bioengineering Institute (China). All other reagents were ultrapure grade.

Monocytes, THP-1, THP-1 MyD88-/- or SupT1 T lymphocytes were plated at a concentration of 1x10⁶ cells per well in 96- or 24-well plates and mock-treated or treated with CLI-095 at 1 μg/ml for 1h. Then cells were stimulated with HPIV3 WT, HPIV3/EboGP, HPIV3/ΔF-HN/EboGP, EBOV GP beads, EBOV at MOI 0.1, 1 or 3 PFU/cell or mock-stimulated, transfected Poly I:C (10 μg/ml), VLPs at 10 μg/ml or 25 μg/ml, CD3/CD28 beads at a ratio of 1 bead per 3 cells, or LPS at 100 or 500 ng/ml and harvested at the indicated time points. Cell were collected at the indicated time points and lysed in RIPA buffer (ThermoFisher Scientific). Proteins were separated by SDS-PAGE using gradient 4–12% gels (ThermoFisher Scientific) and transferred to nitrocellulose membranes (ThermoFisher Scientific) using the I-blot system (ThermoFisher Scientific). Membranes were blocked with 5% milk and 0.1% Tween-20 in PBS for 1 h at 37°C and stained with antibodies specific for the following molecules: TRAM1 (Abcam #ab96106), phosphorylated TRAM1 (FabGennix #PTRAM-140AP), MyD88 (#4283S), IRAK4 (#4363S), phosphorylated IRAK4 (#11927S), Pyk2 (#3090S), phosphorylated Pyk2 (#3291S), p38 (#8690S), phosphorylated p38 (#4511S), NFκB (#6956S), phosphorylated NFκB (#3033S) and GAPDH (#8884S) (all Cell Signaling Technology) diluted according manufacturer’s recommendations in PBS with 0.1% of Tween-20.

Cells were pelleted by centrifugation, washed once with ice-cold PBS, and lysed on ice for 30 min using the Cell Signaling lysis buffer (#9803) according to manufacturer`s extraction protocol. Protein quantitation was done using the Direct Detect system (Millipore). A total of 30 μg of protein was denatured in Laemli buffer at 95°C for 5 minutes and western immunoblotting was performed using the Bio-rad system (TGX 4–15% gels). Transfer was performed using the Trans Blot turbo system (Bio-rad) into PVDF membranes. Images were acquired by using the Bio-rad Imaging Chemidoc MP system. Secondary anti-rabbit and anti-mouse HRP-conjugated antibodies were purchased from Bio-rad (#170-6515, #170-6516). Proteins were detected using the following antibodies purchased from Cell Signaling Technology: Acetyl Histone H3 Lys9 (#9649), Caspase3 (#9668), PARP (#9542), pPRAS40 (#13175), p4EBP1 (#2855), pS6 (#4856), S6 (#2217), BTK (#3533), SYK (#13198), MyD88 (#4283), BCL10 (#4237), IRAK4 (#4363), IkBa (#4812), IKKb (#2370). c-MYC (#32072) was purchased from Abcam. Beta-Actin was from SIGMA (A5316#).

Kwik-Diff stain (9990700) was from Thermo Scientific Shandon. Reagents for priming neutrophils were LPS (E. coli-derived, Sigma, L3024), mouse TNFα (R&D Systems, 410-MT-010), and mouse GM-CSF (Peprotech, 315-03). Reagents for stimulating neutrophils included f-Met-Leu-Phe (fMLP, Sigma, F3506), C5a (Sigma, C5788), and phorbol 12-myristate 13-acetate (PMA, Sigma, P1585). Antibodies for western blotting were Rac1 (clone 23A8, Millipore, 05-389, 1:3000), Rac2 (Millipore, 07-604, 1:5,000), Irak4 (Cell Signaling Technology, 4363, 1:500), MyD88 (Cell Signaling Technology, 4283, 1:500), TLR4 (Cell Signaling Technology, 14358, 1:500), and Trif (Abcam, ab13810, 1:1,000). Antibodies for LPS/TLR4 pathway analysis were from Cell Signaling Technology: phospho-p38 Mapk Thr180/Tyr182 (9211, 1:1000), p38 Mapk (9212, 1:1000), phospho-p42/44 Erk Thr202/Tyr204 (9106, 1:1000), p42/44 Erk (9102, 1:1000), phospho-Akt Thr308 (9275, 1:5000), and Akt (9272, 1:1000). Antibodies for other applications are listed in the relevant sections below. Fc block (553141) was from BD Biosciences.