Anti phospho c jun ser63

RPMI-2650 cells were grown on glass coverslips in 6-well plates for 24 h. Before fixing, cells were pretreated with inhibitors for 1 h, followed by treatment with LPS (10 μg/ml) for 1 h. The treated cells were fixed with 4% formaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS at 4°C for 10 min. After blocking with 3% BSA in PBS at 37°C for 30 min, cells were probed with anti-phospho-c-Jun (Ser63, Cell Signaling) and Alexa Fluor 488-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). DAPI (0.2 μg/ml) was used for nuclear staining. The stained cells were then analyzed under a fluorescence microscope (Carl Zeiss, Göttingen, Germany) with an objective (oil immersion, aperture 1.3) of magnification 63×.

The human PC cell lines, BxPC-3 and Panc-1, were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% dialyzed heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin in a 95% air/5% CO₂ humidified atmosphere at 37°C. Catalase derivative conjugated to polyethylene glycol (PEG-catalase) and streptozotocin (STZ) were acquired from Sigma Aldrich (St. Louis, MO, USA). The ERK inhibitor PD 98059 and the p38 MAPK inhibitor SB 203580 were obtained from Sigma Chemical Co. Primary antibodies against SOD1, SOD2, CAT, GPX1 and uPA were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-ERK, anti-phospho-ERK (Thr202/Tyr204), anti-p38 MAPK, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-NF-κB, anti-phospho-NF-κB p65 (Ser468), anti-c-Jun and anti-phospho-c-Jun (Ser63) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA).

Western blot analysis was performed as previously described [40 (link)]. In brief, the cell lysate was electrophoresed and transferred to a polyvinylidene difluoride (PVDF) sheet (Sigma-Aldrich), then the membranes were nonspecifically blocked in 1% skim milk solution and incubated with the primary antibodies followed by respective HRP-conjugated secondary antibodies. The antibodies used for Western blotting were rabbit monoclonal antibodies against extracellular signal-regulated kinases (ERK)1/2, phospho-ERK1/2 (Thr202/Tyr204), anti-p38, phospho-p38 (Thr180/Tyr182), c-Jun N-terminal kinases (JNK), phospho-JNK (Thr183/Tyr185), anti-c-Jun, anti-phospho-c-Jun (Ser63), and goat-anti-rabbit IgG conjugated with horseradish peroxidase (Cell Signaling Technology, Danvers, MA, USA). Anti-β-actin antibody was used as an internal control (Sigma-Aldrich, St. Louis, MO, USA). Blots were visualized by chemiluminescence reaction (ECL; GE Healthcare, Little Chalfont, UK), and band intensities were measured using Image J (version 1.42; http://rsb.info.nih.gov/ij).

Anti-ERK, anti-JNK, anti-p38α/β (A-12), and anti-HA-probe (Y-11) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-p44/42 MAPK (ERK1/2) (Thr-202/Tyr-204), anti-phospho-SAPK/JNK (specific for phospho-Thr-183 and phospho-Tyr-185), anti-phospho-p38 MAPK (Thr-180/Tyr-182), anti-cleaved PARP (Asp-214), anti-caspase 3, and anti-phospho-c-Jun (Ser-63) antibodies were from Cell Signaling Technology (Danvers, MA). Monoclonal anti-FLAG antibody, anti-FLAG M2 affinity gel, and sodium orthovanadate (Na₃VO₄) were from Sigma-Aldrich (St. Louis, MO). Glutathione Agarose 4B was purchased from Incospharm (Daejeon, Korea).

Cultured FLS (passage 4) were plated at 2 × 10⁵ cells in six-well plates overnight in DMEM containing 10% FBS and synchronized for 24 h in DMEM/0.1% FBS. The cells were pretreated for 1 h with different concentrations of TRYP, TRYP-Ox, or DMSO (vehicle control) and then stimulated with 2 ng/ml IL-1β or medium for 15 min at 37°C. The cells were then washed once with ice-cold PBS and lysed with modified radioimmunoprecipitation assay buffer [50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 2.5 mM MgCl₂, 1.0 mM EDTA, 20 mM β-glycerophosphate, 10 mM NaF, 1 mM Na₃VO₄, and Protease inhibitor cocktail (Roche, Indianapolis, IN)]. Protein concentration of the lysates was measured using a MicroBCA Assay kit (Pierce, Rockford, IL), and 40 μg lysate was subjected to 10% SDS-PAGE and Western blot analysis. Anti–phospho-c-Jun (Ser63) was purchased from Cell Signaling Technology (Danvers, MA), and anti-mouse GAPDH was from Santa Cruz Biotechnology (Santa Cruz, CA).

Hut78 cells were treated with JNK-IN-8 (Med Express) or IL-15 (R&D Systems) for the time indicated. RNA was extracted with a MagMAX Total RNA isolation kit (Invitrogen), reverse transcribed with a SuperScript III First-strand Synthesis system (Invitrogen). Quantitative PCR was performed in technical triplicate using SsoAdvanced Universal SYBR Green (Bio-Rad) and primers purchased from Integrated DNA Technology (Table S4). For western blotting, proteins were extracted in RIPA buffer with 1 mM phenylmethylsulfonyl fluoride (Cell Signaling Technology, 8553) and proteinase/phosphatase inhibitors (Cell Signaling Technology, 5872). Anti-c-Jun (Cell Signaling Technology, 9165; 1:1000), anti-phospho-c-Jun Ser63 (Cell Signaling Technology, 9261; 1:1000) and anti-tubulin (Cell Signaling Technology, 9099; 1:1000) antibodies were used for western blotting.

All cell culture materials were obtained from Gibco Inc. SP600125 and AS601245, pharmacological inhibitors of JNK, were purchased from Calbiochem. Control (#6568) and SAPK/JNK (#6232) siRNA, SAPK/JNK Kinase Assay Kit (#8794, nonradioactive), Hoechst 33342 (#4082), Anti-c-Jun (#9165), Anti-Phospho-c-Jun^Ser63 (#12598), Anti-Phospho-c-Jun^Ser73 (#3270 S), Anti SAPK/JNK (#9252), Anti-Phospho-SAPK/JNK^{Thr183/Tyr185} (#9251), Anti-Cyclin A (4656), Anti-Cyclin B1 (#12231) and Anti-Phospho-cdc-2^Tyr15 (#4539) antibodies were obtained from Cell Signaling. All western blotting buffers and reagents were purchased from Bio-Rad. Anti-phospho-Histone H3^Ser10 (06-570) antibody was obtained from Upstate. Anti-Vinculin Antibody (V9264) was purchased from Sigma-Aldrich. TransIT-X2® Dynamic Delivery System (MIR 6000) was purchased from Mirus Bio LLC. YO-PRO®-1 Iodide (491/509) was obtained from Life Technologies. Texas Red™-X Phalloidin and Click-iT™ EdU Alexa Fluor™ 488 Imaging Kit (C10337) were obtained from Thermo Fisher Scientific. Matrigel Basement Membrane Matrix (356230) was from BD Biosciences. Super Block reagent (#AAA125) was purchased from ScyTek Laboratories.