Phospho mapk family antibody sampler kit

Cells were lysed in RIPA lysis buffer (Beyotime, Shanghai, China). The concentration of each protein sample was measured using a BCA assay kit (Sigma-Aldrich, St. Louis, MO, USA). The samples were separated by SDS-PAGE electrophoresis and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% dehydrated milk for 2 hours and then incubated with the primary antibodies at 4°C overnight. The information of primary antibodies used was as follows: RUNX2 (#12556, Cell Signaling Technology), EVL(#12536, Cell Signaling Technology), mitogen-activated protein kinase (MAPK) Family Antibody Sampler Kit (#9926, Cell Signaling Technology), Phospho-MAPK Family Antibody Sampler Kit (#9910, Cell Signaling Technology), DMP1 (PA5-88069, Invitrogen), DSPP (sc-73632, Santa Cruz), OSX (PA5-115697, Invitrogen), OCN (ab93876, Abcam), and GAPDH (#2118, Cell Signaling Technology). Proteins were quantified using the enhanced chemiluminescence kit (Pierce, IL, USA).

Western blot was done as previously described [19 (link)–21 (link)]. Primary antibodies used in the study were as follows: anti-TRPA1 (1:1000, Alomone), anti-TRPA1 (1:1000, Novus Biologicals, CO, USA), anti-TRPA1 (1:1000, LSBio, WA, USA), MAPK Family Antibody Sampler Kit (1:1000, Cell Signaling Technology), Phospho-MAPK Family Antibody Sampler Kit (1:1000, Cell Signaling Technology), anti-MKP-1 (1:1000, Thermo Fisher Scientific), PGC-1α (1:1000, Abcam), anti-β-actin (1:1000, Abcam), anti-β-tubulin (1:1000, Cell Signaling Technology). Secondary antibodies used were: HRP-conjugated goat anti-rabbit secondary antibody (1:3000, Dako), HRP-conjugated goat anti-mouse secondary antibody (1:3000, Dako).

Antibodies for VEGF, alpha-smooth muscle actin (α-SMA), and Histone H3 were purchased from Abcam (Cambridge, UK). Antibody for CD34 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for Acetyl-Histone H3 (Lys9) (H3K9ace), Ras, Akt GAPDH and Phospho-MAPK family antibody sampler kit, were obtained from Cell Signaling. Dynabeads protein A was obtained from Invitrogen. Supersignal west pico chemiluminescent substrate was purchased from Pierce (Rockford, IL). Truseq RNA sample prep kit-v2, Truseq DNA sample prep kit-PCR box, c-BOT Multiplex re-hybridization plate and Truseq Sbs kit V3 were all purchased from Illumina.

After the indicated treatments, the proteins from the RA FLS were extracted using a cell lysis buffer. The protein concentration was determined with the Bradford assay (Bio-Rad Protein Assay; Bio-Rad, CA, USA). Protein samples (10–20 µg) were resolved in 8% gradient SDS-PAGE, transferred onto PVDF membranes (Merck Millipore, Darmstadt, Germany) and probed with primary antibodies directed against WNT5A (R&D), p44/42, SAPK/JNK, and p38 MAPK (MAPK Family antibody sampler kit, #9926), AKT (#9272), GSK3β(#9315), phospho-p44/42, phospho-SAPK/JNK, and phospho-p38 MAPK (Phospho-MAPK Family antibody sampler kit, #9910), phospho AKT (#9271), phospho GSK-3β (#9322), all from Cell Signaling Technology, Danvers, USA; and GAPDH antibody (Sigma-Aldrich). Bound antibodies were revealed with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling and Santa Cruz Biotechnology), and blots were developed using the ECL Plus detection system (ChemiDoc™ MD Imaging System (Bio-Rad, California, EEUU)).

Acetaminophen, paeonol, sodium carboxymethyl cellulose, N-Acetyl-L-cysteine, hematoxylin and eosin were purchased from Sigma Aldrich (St. Louis, MO, USA). NF-κB pathway antibody sampler kit (#9936), MAPK family antibody sampler kit (#9926), Phospho-MAPK family antibody sampler kit (#9910) and anti-β-actin antibody (#4970) were purchased from Cell Signaling Technology (Beverly, MA, USA). Trizol reagent and SYBR^® Green PCR Master Mix were purchased from Invitrogen (Carlsbad, CA, USA). 2,7-dichlorofluorescein diacetate (DCFH-DA) purchased from Life Technologies (Grand Island, NY).

Whole proteins from the cultured cells were extracted according to the manufacturer’s protocol (RIPA) (Biyuntian, Shanghai, China), followed by concentration investigation. All samples were separated by SDS-PAGE and transferred following standard protocols. Antibodies to CD117 (Abcam, Cambridge, MA, USA) and FcεRI (Abcam) were used to identify mature mast cells. Rho-GTPase Antibody Sampler Kit (Cell Signaling Technology, Danvers, MA, USA), Phospho-MAPK Family Antibody Sampler Kit (Cell Signaling Technology) and Phospho-Stat Antibody Sampler Kit (Cell Signaling Technology), were used to detect the activation of signaling pathways in colon tumor cells. Antibody to cleaved caspase3 (Millipore, Temecula, CA, USA) was used to identify cell apoptosis. Antibody to β-tubulin (Cell Signaling Technology) was used to ensure the consistency of each cell lysate. Antibody to Pseudomonas Exotoxin A (Sigma-Aldrich Corp., St. Louis, MO, USA) and mouse anti-Human IgE antibody (Abcam) were used to identify the recombinant protein toxin. HRP-conjugated secondary antibodies were purchased from Cell Signaling and SuperSignal chemiluminescent reagent was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Protein expression was evaluated using Quantity One software according to immunoblotting band intensity.

The anti-His-tagged, anti-IκBα, anti-IL-1β, anti-PCNA, MAPK Family Antibody Sampler Kit and Phospho-MAPK Family Antibody Sampler Kit were supplied by Cell Signaling Technology (Beverly, MA, USA). Antibodies against p65 and actinin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). BMS-345541, TPCA-1, SC-514, and SP600125 were purchased from Selleckchem. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Isopropyl β-d-1-thiogalactopyranoside (IPTG) and imidazole were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Human Inflammatory Response and Autoimmunity RT² Profiler™ PCR Array (PAHS-077ZA-6) was purchased from Qiagen (Valencia, CA, USA). All human and mouse cytokines ELISA kits were obtained from eBioscience (San Diego, CA, USA). Zeba™ spin desalting columns, HisPur Ni-NTA superflow agarose, high-capacity endotoxin removal spin columns and LAL chromogenic endotoxin quantitation kit were purchased from Thermo Scientific (Rockford, IL, USA). Rosetta™ 2 (DE3) pLysS competent cells were obtained from EMD Millipore Corporation (Billerica, MA, USA).