Anti p jnk

Total proteins of BMMSCs were isolated using RIPA (Cell Signaling Technology, USA). The automated western blot was performed using Simple Wes (Protein Simple, USA) following the manufacturer’s protocol. Briefly, 2.5 μg of protein from the cell lysates was added to the standard fluorescent mastermix, then was loaded into corresponding wells of the prefilled Wes assay plate, along with antibody diluent (Protein Simple, USA), anti-MagT1 (Proteintech, USA), anti-DSP (Santa Cruz, USA), anti-DMP-1 (Genetex, USA), anti-ALP (Affinity, USA), anti-ERK (Cell Signaling Technology, USA), anti-p-ERK (Cell Signaling Technology, USA), anti-JNK (Cell Signaling Technology, USA), anti-p-JNK (Cell Signaling Technology, USA), anti-p38 (Cell Signaling Technology, USA), anti-p-p38(Cell Signaling Technology, USA), anti-β-Tublin (Affinity, USA), anti-rabbit secondary antibody (Protein Simple, USA), and Streptavidin-HRP, followed by luminal peroxide mix. The imaging and analysis were done with compass software (Protein Simple).

In this study, the antibodies respectively against p38, p-p38, ERK, p-ERK, Akt1/2/3, p-Akt1/2/3, OGT, OGA, CD68 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal Anti-β-O-Linked N-Acetylglucosamine Clone CTD110.6 was obtained from Sigma (St. Louis, MO, USA). Other antibodies including anti-JNK, anti-p-JNK, Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA).
Chemicals such as 1-phenyl-3-methyl-5-pyrazolone (PMP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Tris, Tween 20, trifluoroacetic acid, LPS (from Escherichia coli 055:B5), 6-diazo-5-oxo-L-norleucine (DON) and bovine serum albumin were obtained from Sigma. The ABC kit and Agarose wheat germ agglutinin (WGA) were purchased from Vector Laboratories (Lowellville, OH, USA). Thiamet G (Thi G) was obtained from Selleck Chemicals (Houston, TX, USA). RPMI 1640 was purchased from Corning Incorporated (Corning, NY, USA), penicillin, streptomycin and heat-inactivated fetal bovine serum (FBS) was from Gibco (Grand Island, NY, USA). All the other chemicals including sodium dodecyl sulfonate, ammonium persulfate, isopropanol, hydrochloric acid, glycine, sodium chloride and ammonia were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

MEPMs were serum starved overnight and treated as for RNA-seq and then washed 3× in ice-cold PBS (Phosphate-buffered saline) before being harvested in NP-40 lysis buffer (20 mM Tris–HCl pH 8, 150 mM NaCl, 10%glycerol, 1% Nonidet P-40, 2 mM EDTA (Ethylenediaminetetraacetic acid), 1× complete Mini protease inhibitor cocktail [Roche Applied Science, Indianapolis, IN], 1 mM PMSF (Phenylmethanesulfonylfluoride), 10 mM NaF, 1 mM Na3VO4, and 25 mM β-glycerophosphate). Total cell lysates were sonicated briefly and then collected by centrifugation. Lysates were then resuspended in Laemmli buffer containing 10% β-mercaptoethanol, heated at 95°C for 5 min, and separated by SDS-polyacrylamide gel electrophoresis.
The following inhibitors were used: LY294002 (Sigma-Aldrich), PD325901 (Stemgent, Cambridge, MA), cycloheximide (Fisher Scientific, Waltham, MA), and Bim I (Santa Cruz Biotechnology, Dallas, TX).
The following antibodies were used: anti-phospho MAPK p42/p44 (9201; Cell Signaling Technologies, Danvers, MA; 1:1000), anti-pAkt (9271; Cell Signaling Technologies; 1:1000), and anti-pJNK (4671; Cell Signaling Technologies; 1:1000). The anti-β tubulin E7 antibody (1:1000) developed by M. Klymkowsky was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA.

Cells were lysed and sonicated in radioimmunoprecipitation (RIPA) buffer with protease inhibitor mixture, PMSF, and sodium orthovanadate. Protein concentration was measured by BCA protein assay (Thermo scientific, Rockford, IL, USA). The samples were resolved by SDS-PAGE and transferred to nitrocellulose membrane and blotted with specific antibodies: anti-p58^IPK, anti-p-NF-κB, anti-NF-κB, anti-p-p38, anti-p38, anti-p-JNK, anti-JNK, (Cell Signaling Technology, Boston, MA, USA), anti-p-PKR, anti-PKR, anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-IL-1β (R&D Systems, Inc., Minneapolis, MN, USA), anti-caspase-1 (p-20), and anti-NLRP3 (AdipoGen, Inc, San Diego, CA, USA). The immunoblots were developed using chemiluminescence (SuperSignal West Dura Extended Duration Substrate, Thermo Scientific, USA) and visualized under Chemidoc MP Imaging System (Bio-Rad, Hercules, CA, USA).

Total protein was extracted from CRC tissues and cultured cells and subjected to immunoblotting, as previously described [20 (link)]. Primary antibodies including rabbit polyclonal anti-claudin-1 (1:1000), anti-claudin-2 (1:500), anti-claudin-3 (1:1000), and anti-claudin-7 (1:1000) were obtained from Abcam (Cambridge, MA, USA). Primary antibody rabbit polyclonal anti-occludin (1:400) was purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies including rabbit polyclonal anti-E-cadherin (1:1000), anti-c-kit (1:1000), anti-c-Jun (1:1000), anti-p-c-Jun (1:1000), anti-JNK (1:1000), and anti-p-JNK (1:1000) were obtained from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal anti-β-actin (1:2000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The proteins were detected using enhanced chemiluminescence (ECL) (ThermoFisher Scientific, Waltham, MA, USA) and viewed in Fusion FX Vilber Lourmat (Paris, France).

Antibodies and reagents used for flow cytometry were: CD8-APC, Fixable viability stain 450, antiCD16/32 (eBiosciences); anti-rabbit-PE (Jackson Immunoresearch); anti-SLP76 (pY128), CD4-PE, CD25-FITC, CD8 FITC and anti-mouse IgG1-PE (BD Biosciences); CD4-PEVio770 (Miltenyi Biotec); CD3-BV421 (Biolegend); anti-pErk1/2 (pT202/pY204) (Cell signaling Technology). The following reagents were used for cell stimulation, immunoblotting or immunoprecipitation: anti-CD3-biontinylated, anti-CD28-biontinylated, anti-CD3ε, anti-CD28 (eBiosciences); streptavidin (Sigma); anti-pErk1/2 (pT202/pY204), anti-pPLCγ1 (pY783), anti-pp38 (pT180/pY182), anti-pJNK (pT183/Y185), anti-pAkt (pS473), anti-pSLP76 (pS376) (Cell Signaling Technology); anti-GADS (Santa Cruz Biotechnology Inc.); rabbit anti-SLP76 or goat-anti-SLP76 (Thermo Fisher Scientific); anti-β-tubulin (Chemicon); for anti-14-3-3 immunoblotting we used a mix of anti-pan 14-3-3 and anti-14-3-3ζ (Santa Cruz, Biotechnology Inc.).