Phospho sapk jnk thr183 tyr185

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Phospho-SAPK/JNK (Thr183/Tyr185) is a lab equipment product that detects phosphorylation of SAPK/JNK at Thr183 and Tyr185.

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Phospho-JNK (453 citations) SAPK/JNK (140 citations) Phospho-SAPK/JNK (78 citations) Phospho-JNK (Thr183/Tyr185) (40 citations) Anti-phospho-SAPK/JNK (Thr183/Tyr185) (20 citations) JNK/phospho-JNK (6 citations) SAPK)/JNK (Thr183/Tyr185), (5 citations) Rabbit anti-phospho-SAPK/JNK (Thr183/Tyr185, (5 citations) Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) (5 citations) PathScan® Phospho-SAPK/JNK (Thr183/Tyr185) Sandwich ELISA Kit (5 citations)

44 protocols using phospho sapk jnk thr183 tyr185

Signaling Pathway Antibody Validation

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Antibodies used were phospho-IkBa (Ser32/36) (9246, Cell Signaling), phospho-SAPK/JNK (Thr183/Tyr185) (9251, Cell Signaling), SAPK/JNK (9252, Cell Signaling), IkBa (SC-371, Santa Cruz), phospho-IKKα/β (Ser176/180) (2697, Cell Signaling), Phospho-p38 MAPK (Thr180/Tyr182) (9215, Cell Signaling), Phospho-NF-κB p65 (Ser536) (3033, Cell Signaling), tubulin (ab7291, Abcam), actin (AAN01-A, Cytoskeleton), BiP (610978, BD Biosciences), CHOP (MA1-250, ThermoFisher), SLC39A7 (19429-1-AP, Proteintech), TNFR1 (sc-8436, Santa Cruz), TRAIL-R1/DR4 (42533, Cell Signaling), RIPK1 (610458, BD Bioscience), RIPK3 (12107, Cell Signaling), GFP (sc-69779, Santa Cruz), FADD (610399, BD Biosciences), phospho-MLKL (Ser385) (ab187091, Abcam), phospho-RIPK1 (S166) (65746, Cell Signaling), phospho-RIPK3 (S227) (ab209384, abcam), cleaved Caspase-3 (Asp175) (9661, Cell Signaling), V5 (R960-25, Invitrogen or ab9116, abcam), PDIA2 (ab2792, abcam), TNIP1/ABIN-1 (4664, Cell Signaling), cIAP1/BIRC2 (7065, Cell Signaling), cIAP2/BIRC3 (3130, Cell Signaling) and LAMP1 (ab25630, abcam). The secondary antibodies used were goat anti-mouse HRP (115-035-003, Jackson ImmunoResearch), goat anti-rabbit HRP (111-035-003, Jackson ImmunoResearch), Alexa Fluor 680 goat anti-mouse (A-21057, Molecular probes) and IRDye 800 donkey anti-rabbit (611-732-127, Rockland).

Fauster A., Rebsamen M., Willmann K.L., César-Razquin A., Girardi E., Bigenzahn J.W., Schischlik F., Scorzoni S., Bruckner M., Konecka J., Hörmann K., Heinz L.X., Boztug K, & Superti-Furga G. (2018). Systematic genetic mapping of necroptosis identifies SLC39A7 as modulator of death receptor trafficking. Cell Death and Differentiation, 26(6), 1138-1155.

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Immunoblotting Analysis of Apoptosis and Signaling Pathways

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Antibodies used were: cleaved caspase-3 (#9661), cleaved poly (ADP-ribose) polymerase (#9541), AKT-total (#4691), phospho-AKT(Ser473) (#4060), JAK2 (#9945S), phospho-SAPK/JNK (Thr183/Tyr185) (#6251S), STAT3 (#9139S), phospho-STAT3 (Tyr705) (9145S) and phospho-STAT3 (Ser 727), E-cadherin (#4065) (9134) (Cell Signaling, Beverly, MA); Flag M2 (F3165), Vimentin (V6630), DLEC1 (HPA019077) and β-actin (AC-74) (Sigma-Aldrich, St. Louis, MO); anti-mouse Ig G-HRP (P0161), anti-rabbit Ig G-HRP (P0448) (Dako ,Glostrup, Denmark); Twist (sc-15393; Santa Cruz, CA, USA); a-tubulin (Lab Vision Corporation, Fremont, CA); V5-Tag (MCA1360; AbD Serotec, Raleigh, NC).
Human IL-6 (PF01229) (Peprotech, Rocky Hill, NJ) was used. Western blot and IP experiments were performed according to previous protocols 48 (link), 49 (link).

Li L., Xu J., Qiu G., Ying J., Du Z., Xiang T., Wong K.Y., Srivastava G., Zhu X.F., Mok T.S., Chan A.T., Chan F.K., Ambinder R.F, & Tao Q. (2018). Epigenomic characterization of a p53-regulated 3p22.2 tumor suppressor that inhibits STAT3 phosphorylation via protein docking and is frequently methylated in esophageal and other carcinomas. Theranostics, 8(1), 61-77.

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Linarin Extract from Flos Chrysanthemi

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Linarin extract was processed from Flos Chrysanthemi (Zhejiang Chinese Medical University, Traditional Chinese Medicine Decoction Pieces, Ltd). The amount of linarin was determined by HPLC analysis as described in our previous protocol [19 (link)]. The linarin content in the extract is 72%. HFHC diet (10% fat, 1% cholesterol, 0.2% cholate) were prepared by TROPHIC Animal Feed High Tech Co. (Nantong, China). Kits for serum biochemistry analysis including cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol, (HDL-c), low density lipoprotein-cholesterol (LDL-c), glucose and alanine transaminases (ALT), and aspartate transaminases (AST) were purchased from MeiKang Chemical Co. (Ningbo, China). Liver TC and TG quantification kits were from Applygen Technologies Inc. (Beijing, China). Both First-strand cDNA synthesis kit and FastStart Universal SYBR Green Master (ROX) kit were products from Roche (USA). BCA kit for protein quantification was from Beyotime Biotechnology (Shanghai, China). Phospho-SAPK/JNK (Thr183/Tyr185) and SAPK/JNK primary antibody were purchased from Cell Signaling Technology (USA).

Zhuang Z.J., Shan C.W., Li B., Pang M.X., Wang H., Luo Y., Liu Y.L., Song Y., Wang N.N., Chen S.H., Shi J.P, & Lv G.Y. (2017). Linarin Enriched Extract Attenuates Liver Injury and Inflammation Induced by High-Fat High-Cholesterol Diet in Rats. Evidence-based Complementary and Alternative Medicine : eCAM, 2017, 4701570.

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LPS and 5-HMF Induced Cell Signaling

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LPS (Escherichia coli O111:B4) and 5-HMF were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Gibco (ThermoFisher, Gaisburg, MD, USA). Fetal bovine serum (FBS) was purchased from Lonsera (Lonsa Science SRL, Guichon, Uruguay). MTT cell proliferation and cytotoxicity assay kit was purchased from Phygene life sciences (Fuzhou, China). The primary rabbit monoclonal antibodies against p38 MAPK (D13E1) XP^®, SAPK/JNK Antibody, p44/42 MAPK (ERK1/2) (137F5), phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP^®, phospho-SAPK/JNK (Thr183/Tyr185) (81E11), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E) XP^®, Akt (pan) (C67E7), mTOR (7C10), phospho-Akt(Ser473) (D9E) XP^®, phospho-mTOR (Ser2448) (D9C2) XP^®, IκBα (44D4), NF-κB p65 (D14E12) XP^®, phospho-IκBα (Ser32) (14D4) and phospho-NF-κB p65 (Ser536) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Kong F., Lee B.H, & Wei K. (2019). 5-Hydroxymethylfurfural Mitigates Lipopolysaccharide-Stimulated Inflammation via Suppression of MAPK, NF-κB and mTOR Activation in RAW 264.7 Cells. Molecules, 24(2), 275.

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Detecting SAPK/JNK Activation in Cells

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For detection of pSAPK/JNK proteins, cells were seeded into a 6-well dish at 1x10⁶ cell per well and allow to rest overnight at 37°C in 5%CO2 incubator. LGG was grown as described previously. Cells were treated with LGG, Sham, LPS 100ng/ml, or UV light for the indicated amount of time. Cell monolayers were washed twice with PBS and whole cell lysates were obtained using RIPA buffer containing protease and phosphatase inhibitors (Cell Signaling Technology, 5872). Proteins were resolved in 5–20% gradient SDS-PAGE, then transferred to PVDF membranes for western blotting. Antibodies directed against the following proteins were used: Phospho-SAPK/JNK (Thr183/Tyr185) (Cell Signaling Technology, 4668), SAPK/JNK (Cell Signaling Technology, 9252) and Histone H3 (Cell Signaling Technology, 4499). Goat-anti-rabbit horseradish peroxidase conjugated secondary antibody was used for indirect detection of target proteins.

Kumova O.K., Fike A.J., Thayer J.L., Nguyen L.T., Mell J.C., Pascasio J., Stairiker C., Leon L.G., Katsikis P.D, & Carey A.J. (2019). Lung transcriptional unresponsiveness and loss of early influenza virus control in infected neonates is prevented by intranasal Lactobacillus rhamnosus GG. PLoS Pathogens, 15(10), e1008072.

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Antibody Reagents for Stem Cell Research

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Antibodies against Sox2 (#3579), Nanog (#4903), Bmi1 (#6964), E-cadherin (#3195, for immunocytochemistry), glucocorticoid receptor (#12041), phospho-glucocorticoid receptor (Ser-211) (#4161), c-Jun (#9165), phospho-c-Jun (Ser-63) (#9261), phospho-SAPK/JNK (Thr-183/Tyr-185) (#4668), survivin (#2808), GFAP (#3670), phospho-Smad2 (Ser-465/467) (#3108), Smad2/3 (#8685), and GAPDH (#5174) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies against JNK1 (sc-474), JNK2 (sc-7345), E-cadherin (sc-8426, for immunoblotting), and MKP-1 (sc-370) were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The antibody against nestin (MAB5326) was purchased from Merck Millipore (Darmstadt, Germany). Anti-CD133 (W6B3C1) was purchased from Miltenyi Biotech (Bergisch Gladbach, Germany). Anti-β-actin (A1978) was from Sigma–Aldrich. Dexamethasone (DEX) was purchased from Fuji Pharma Co., Ltd. (Tokyo, Japan). Prednisolone (PSL), 5-FU, and gemcitabine (GEM) were from Sigma–Aldrich. DEX, PSL, 5-FU, and GEM were dissolved in PBS (for DEX and PSL) or DMSO (for 5-FU and GEM) to prepare 1, 10, 200, and 1 mm stock solutions for in vitro use, respectively.

Suzuki S., Okada M., Sanomachi T., Togashi K., Seino S., Sato A., Yamamoto M, & Kitanaka C. (2021). Therapeutic targeting of pancreatic cancer stem cells by dexamethasone modulation of the MKP-1–JNK axis. The Journal of Biological Chemistry, 295(52), 18328-18342.

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Protein Expression Analysis via Western Blot

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Western blot was performed as previously reported40 (link)46 (link)47 (link)48 (link). The Phospho-MEK1/2 (Ser217/221) (#9154), ERK1/2 (#4695), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370), JNK/SAPK (#9258), Phospho-SAPK/JNK (Thr183/Tyr185) (#9255), p38 MAPK (#8690), Phospho-p38 MAPK (Thr180/Tyr182) (#4511), and β-actin (#8457) antibodies were purchased from Cell Signaling Technology. Antibodies against APOA4 (D221656), CKM (D260075), CA2 (D120344), TNNI3 (D120341), MYL3 (D122718), CRP (D120482), FASN (D262701), GLUL (D122427), A2M (D162821), and PLG (D262067) were obtained from BBI Life Sciences. All the primary antibodies were diluted as 1:1000. The protein signals were detected by using a ChemiDoc XRS+ Imaging System (Bio-Rad), and then the gray value of proteins were analyzed by Image lab software (version 5.2.1, Bio-Rad).

Jiang D.S., Zeng H.L., Li R., Huo B., Su Y.S., Fang J., Yang Q., Liu L.G., Hu M., Cheng C., Zhu X.H., Yi X, & Wei X. (2017). Aberrant Epicardial Adipose Tissue Extracellular Matrix Remodeling in Patients with Severe Ischemic Cardiomyopathy: Insight from Comparative Quantitative Proteomics. Scientific Reports, 7, 43787.

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Osteoblast and Osteoclast Culture Techniques

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In vitro primary osteoblast cultures, osteoclast cultures, quantitative PCR, Western blotting, ALP and Von Kossa were performed as previously described.^{(11 (link))} Primary antibodies were specific for phospho-JUN, (1:1,000; Cell Signaling) and GAPDH (1:2,000; Sigma), Phospho-SAPK/JNK (Thr183/Tyr185), and SAPK/JNK Antibody (#9251 and #9252 Cell Signaling). Osteoclast culture and RNA isolation from whole bone was performed as previously described.^{(13 (link))} Osteocyte expression analysis was performed in the IDG-SW3 cell line, a generous gift form Dr. Lynda Bonewald.^{(12 (link))}

Xu R., Zhang C., Shin D.Y., Kim J.M., Lalani S., Li N., Yang Y.S., Liu Y., Eiseman M., Davis R.J., Shim J.H, & Greenblatt M.B. (2017). c-Jun N-terminal kinases (JNKs) are critical mediators of osteoblast activity in vivo. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 32(9), 1811-1815.

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Immunofluorescence Staining of IL-18R1, pJNK, and p-c-Jun

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Slides were cleared using Histoclear and rehydrated in stepwise alcohol. Following washing, slides were boiled in citrate antigen retrieval buffer for 20 minutes. After permeabilization with 0.5% triton X-100 in TBS with 2% BSA followed by washing in 1% BSA, slides were incubated with primary antibody at 4°C overnight. Secondary labeling was performed with species-appropriate fluorescent conjugate for 1 hour at room temperature the next day. DAPI was used to label nuclei, and slides were mounted in anti-fade media. Imaging was performed on a Leica confocal microscope. Unstained, primary antibody–only and secondary antibody–only slides were prepared from each patient for comparison. Quantification of fluorescent staining was accomplished using the ImageJ software (version 1.51w). Anti–IL-18R1 antibody was obtained from Sigma-Aldrich (HPA007615; anti–IL-18R1 antibody produced in rabbit). Anti-phJNK antibody was obtained from Cell Signaling Technology (phospho-SAPK/JNK [Thr183/Tyr185], catalog G9; mouse mAb, catalog 9255). Anti–ph–c-Jun antibody was obtained from Cell Signaling Technology (ph–c-Jun [Ser73], catalog 9164).

Camiolo M.J., Zhou X., Wei Q., Trejo Bittar H.E., Kaminski N., Ray A, & Wenzel S.E. (2021). Machine learning implicates the IL-18 signaling axis in severe asthma. JCI Insight, 6(21), e149945.

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Protein Isolation and Western Blot Analysis

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For the protein isolation, 1 × 10⁶ cells were cultured on 100mm Petri dishes and treated with experimental medium containing of DON for 24 h. The protein isolation and Western blots were conducted as previously described [48 (link)]. 30 µg (60 µg for phospho-p44/42 (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182), phospho-SAPK/JNK (Thr183/Tyr185) antibody) of protein samples was used for electrophoresis. SOD1 (#4266), SOD2 (#13141), p65 (#8242), p44/42 (#4695), SAPK/JNK (#9252), p38 (#8690), phospho-p44/42 (Thr202/Tyr204) (#4370), phospho-p38 (Thr180/Tyr182) (#4511), phospho-SAPK/JNK (Thr183/Tyr185) (#4668) (Cell Signaling Technology, Leiden, WZ, The Netherlands) antibodies were used according to manufacturer’s recommendations. The results were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-59540) (Santa Cruz Biotechnology Inc, Dallas, TX, USA) as a reference protein.

Habrowska-Górczyńska D.E., Kowalska K., Urbanek K.A., Domińska K., Sakowicz A, & Piastowska-Ciesielska A.W. (2019). Deoxynivalenol Modulates the Viability, ROS Production and Apoptosis in Prostate Cancer Cells. Toxins, 11(5), 265.

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