P syk

BMDCs were washed and resuspended at 6 × 10⁶cells/mL in RPMI‐1640 with L‐glutamine supplemented with β‐mercaptoethanol (50 μM), penicillin/streptomycin (100 μg/mL) for 3 h prior to stimulation with HKCA (6.25 × 10⁵cells/mL) or curdlan (100 μg/mL) at 37°C for 0–20 min. Cells were lysed (1% Triton, 120 mM NaCl, 50 mM Tris, 0.1% SDS, 1 mM EDTA, containing protease/phosphatase inhibitors) and resolved in SDS‐PAGE gels and transferred to PVDF membranes, blocked (Tris, 5% BSA, 0.05% Tween20) and probed with the indicated antibodies; pSyk (clone; C87C1), Syk (clone; D1I5Q), pErk and Erk (mAb Rabbit IgG), Pp38 (clone; D3F9), p38 (clone; D13E1), IκBα (clone; 44D4), pIκBα (clone; 14D4) (all immunoblotting antibodies from Cell Signaling Technologies) followed by anti‐rabbit‐HRP (Dako) secondary antibody. Proteins visualized by SuperSignal chemiluminescent reaction (Pierce) in a ChemiDoc station (BioRad). Densitometry measurements were performed with ImageJ software.

Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (penicillin and streptomycin), and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) was obtained from Biowest (Kansas City, MO, USA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), LPS, 4-nitrophenyl n-acetyl-b-d-glucosaminide (p-NAG), and monoclonal anti-DNP-IgE were supplied by Sigma–Aldrich (St. Louis, MO, USA). DNP-BSA was procured from Invitrogen (Gaithersburg, MD, USA). Primary antibodies against p-p38, p38, p-JNK, JNK, p-ERK, ERK, p-Lyn, Lyn, p-Syk, Syk, p-PLCγ, PLCγ, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA), and FcεRIγ, from LSBio (Seattle, WA, USA). SensiFAST SYBR No-ROX kit mix was purchased from Biolines (Seoul, Korea), and human filaggrin, AQP3, and HA ELISA kits were obtained from CUSABIO (Seoul, Korea). Rat basophilic leukemia (RBL-2H3, ATCC^® CRL-2256) and murine macrophage (RAW264.7, ATCC^® TIB-71) cells were procured from the American Type Culture Collection (ATCC), while human immortalized keratinocyte (HaCaT) cells were obtained from Prof. Lee of Chosun University in Korea.

The study included 62 DLBCL samples in two tissue microarrays from 2000–2011. Twenty-five cases were from nodal sites and 37 from extranodal sites. Only tumors with enough tissues were included. Immunohistochemical stains of p-STAT3 (Y705, rabbit monoclonal Ab, clone D3A7, Cell Signaling) and p-SYK (Y525/526; rabbit polyclonal Ab; Cell Signaling) were performed and the results were evaluated by proportion of lymphoma cells that were stained. The antibody reactivity in ≥ 30% of the lymphoma cells was considered positive. Paraffin-embedded normal tonsil tissue was included as a baseline expression level for p-STAT3 and p-SYK.

Cr-ME was purchased from the Plant Diversity Research Center (DaeJeon, South Korea). Sodium dodecyl sulfate (SDS), 3-(4,5-dimethylthiazol,2-yl)-2,5-diphenyltetrazolium bromide (MTT), polyethylenimine (PEI), dimethyl sulfoxide (DMSO), polyinosinic: polycytidylic acid (Poly (I:C), and lipopolysaccharide (LPS, Escherichia coli O111:B4) were obtained from Sigma Chemicals Co. (St. Louis, MO, USA). Cell culture chemicals such as fetal bovine serum (FBS) was purchased from Biotechnics Research (Lake Forest, CA, USA), and Roswell Park Memorial Institute 1640 (RPMI 1640) and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from HyClone (Grand Island, NY, USA). The RAW264.7 cell line (No. TIB-71) and human embryonic kidney cell line (HEK293T) (No. CRL-3216) were acquired from the American Type Culture Collection (ATCC, Rockville, MD, USA). Luciferase constructs with NF-κB and the PDR (III-I) luciferase promoter were used as previously reported [53 (link)]. Phospho- and total antibodies against p50, p65, Src, p-Src, Syk, p-Syk, IκBα, p-IκBα, IRF3, TBK1, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Total protein from cells and kidney were isolated by RIPA buffer for western blot analysis as previously described 8 (link)-11 (link), 35 (link). Primary antibodies against NPY, Y1R, phospho (p)-NF-κB/p-p65, NF-κB/p65, p-Syk (all from Cell Signaling), iNOS (Abcam, Cambridge, UK), Mincle (Santa Cruz or MBL International, Woburn, MA), and β-actin (Santa Cruz) were used. After being stained and washed, the membranes were incubated with LI-COR IRDye 800 conjugated secondary antibodies (Rockland Immunochemicals, Gilbertsville, PA) in the dark for 1 hour. Signals were then scanned and visualized by the Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE). The ratio of the targeted protein was subjected to β-actin and quantified with ImageJ software (NIH, Bethesda, MD).