Anti jak2

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

Anti-JAK2 is a primary antibody product manufactured by Cell Signaling Technology. It is designed to detect the Janus Kinase 2 (JAK2) protein, which plays a crucial role in cellular signaling pathways.

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87 protocols using anti jak2

Western Blotting Analysis of Apoptosis Markers

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Cell lysates were prepared by resuspending cell pellets in 1× Laemmli sample buffer containing 5% β-mercaptoethanol. Protein from lung tissues were extracted using IPH lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% NP40) containing protease inhibitor cocktail (Roche). The protein lysates were separated by SDS–PAGE and then electrotransferred to nitrocellulose membranes (Millipore Corp., Bradford, MA, USA). Detection of specific proteins was carried out with enhanced chemiluminescence reagents following the manufacturer’s instructions (P90720, Millipore Corporation, MA, USA). The primary antibodies used for western blotting were as follows: anti-Caspase-9 (#9508), anti-Caspase-8 (#9746), anti-Caspase-7 (#8438), anti-Caspase-3 (#9664), anti-Bim (#2933), anti-Bak (#3814), anti-Bcl-xL (#2762), anti-p-Jak2 (#3771), anti-Jak2 (#3230), and anti-p-STAT3^Y705 (#9145), which were purchased from Cell Signaling Technology. Anti-CDK2 (sc-6248), anti-p53 (sc-126), anti-STAT3 (sc-8019), anti-PARP-1 (sc-74470), anti-Cyclin D1 (sc-8396), and anti-β-actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology. An anti-p21 (ab7960), anti-p16 (ab108349), anti-IL-6 (ab6672), and anti-TNF-α (ab6671) antibodies were purchased from Abcam. Densitometry analyses were performed using ImageJ software (National Institutes of Health, NIH).

Cho H.J., Hwang J.A., Yang E.J., Kim E.C., Kim J.R., Kim S.Y., Kim Y.Z., Park S.C, & Lee Y.S. (2022). Nintedanib induces senolytic effect via STAT3 inhibition. Cell Death & Disease, 13(9), 760.

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Western Blot Analysis of SOCS Proteins

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Adherent cells were lysed in cell lysis buffer, and Western blot analysis was performed as previously described (8 (link)). Cells were treated with the proteasome inhibitor MG132 (N-carbobenzyloxy-L-leucyl-L-leucyl-L-leucinal) (5 μM) for 16 hours before the lysis of cells for the analysis of SOCS4 and SOCS5 proteins. Antibodies used for Western blotting were as follows: anti-pSTAT3 (Y705), total STAT3, pERK (p44/42, T202/Y204), pAKT (S473), total AKT, pS6 (S240/244), S6, pEGFR (Y1068), total EGFR, L858R-EGFR– or del19-EGFR–specific antibodies, anti-JAK2 (Cell Signaling Technology), and anti-JAK1 (BD Biosciences); anti–total ERK, EGFR, Myc, ubiquitin and horseradish peroxidase (HRP)–conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG, HRP-conjugated anti-rabbit IgG, SOCS4, and SOCS5 (Santa Cruz Biotechnology); anti–c-MET (Invitrogen); and anti–α-tubulin (Sigma-Aldrich). Densitometric quantification of high-resolution immunoblot images was performed using an analysis program, ImageJ (developed by W. Rasband, Research Services Branch of the National Institute of Mental Health). Analyses of the phosphorylation status of RTK and ErbB family members were performed using the RayBio Human RTK and EGFR Phosphorylation Array kits according to the manufacturer’s instruction (RayBiotech).

Gao S.P., Chang Q., Mao N., Daly L.A., Vogel R., Chan T., Liu S.H., Bournazou E., Schori E., Zhang H., Brewer M.R., Pao W., Morris L., Ladanyi M., Arcila M., Manova-Todorova K., de Stanchina E., Norton L., Levine R.L., Altan-Bonnet G., Solit D., Zinda M., Huszar D., Lyden D, & Bromberg J.F. (2016). JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors. Science signaling, 9(421), ra33.

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Immunoblotting Analysis of Intestinal Signaling

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Mouse intestinal mucosa, including most proximal and distal regions, was collected by scraping as previously described [27] (link). Mouse epithelial cells were broken in a lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0, 0.2 mM sodium orthovanadate, and protease inhibitor cocktail) (Roche, Nutley, NJ). Immunoblotting was performed with primary antibodies: anti–phospho-Stat1, anti–phospho-Stat3, anti–phospho-Jak2, anti-Stat1, anti-Stat3, and anti-Jak2 (Cell Signal, Beverly, MA), or anti–beta-actin (Sigma-Aldrich, Milwaukee, WI) antibodies and secondary antibodies visualized by ECL [42] (link).

Lu R., Wu S., Zhang Y.G., Xia Y., Zhou Z., Kato I., Dong H., Bissonnette M, & Sun J. (2016). Salmonella Protein AvrA Activates the STAT3 Signaling Pathway in Colon Cancer. Neoplasia (New York, N.Y.), 18(5), 307-316.

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Western Blot Analysis of JAK2 Signaling

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Leukemic cells were lysed in lysis buffer supplemented with freshly added protease and phosphatase inhibitors. 25μg (BCA method; Thermo Scientific) lysate was loaded on 10% mini protean precast gels (BioRad, Veenendaal, Netherlands), and transferred to a nitrocellulose membrane (Biorad). Primary antibody incubation was performed according to manufacturer's protocol. Anti-JAK2 (#3230), anti-phospho-JAK2-Tyr1007 (#4406), anti-phospho-STAT5-Tyr694 (#9351), anti-phospho-STAT1-Tyr701 (#9167), anti-Stat1 (#9175), anti-phospho-MEK1/2-Ser217/221 (#9154), anti-MEK1/2 (#4694), anti-phospho-Erk1/2-T202/204 (#4370), anti-Erk1/2 (#91078), and anti-αTubulin (#2144) were obtained from Cell Signaling Technology (Danvers, Massachusetts, USA). Anti-β-actin (ab6276) was obtained from Abcam (Cambridge, UK), and anti-STAT5 (sc-835) from Santa Cruz (Heidelberg, Germany). Blots were stained with secondary antibodies (IRDye 680RD- or 800CW-labelled anti-rabbit and IRDye 680RD- or 800CW-labelled anti-mouse; Li-Cor Biosciences, Leusden, Netherlands) and scanned using the Odyssey infrared imaging system (Li-Cor Biosciences). To reprobe membranes, they were stripped in NewBlot Nitrocellulose stripping buffer (Li-Cor Biosciences) according to manufacturer's protocol. BCR-JAK2, PAX5-JAK2 and TERF2-JAK2 proteins were separated from wildtype JAK2 based on size (~94, 57, 95 and 125 kDa, respectively).

Steeghs E.M., Jerchel I.S., de Goffau-Nobel W., Hoogkamer A.Q., Boer J.M., Boeree A., van de Ven C., Koudijs M.J., Besselink N.J., de Groot-Kruseman H.A., Zwaan C.M., Horstmann M.A., Pieters R, & den Boer M.L. (2017). JAK2 aberrations in childhood B-cell precursor acute lymphoblastic leukemia. Oncotarget, 8(52), 89923-89938.

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Metformin and TRAIL-Induced Apoptosis

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Metformin was purchased from Wako (Richmond, VA, USA). TRAIL (Recombinant human) was purchased from Millipore (Millipore, Darmstadt, Germany.) Protein G PLUS-Agarose, Anti-Bax, anti-Bcl-2, anti-Mcl-1(IP), anti-Ub and anti-Bcl-xL were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-XIAP, anti-phospho ERK, anti-ERK, anti-phospho JAK2, anti-JAK2, anti-phospho AMPK, anti-AMPK, anti-phospho mTOR, anti-mTOR, anti-phospho AKT, anti-AKT, anti-phsopho GSK3β, anti-GSK3β, anti-Noxa, anti-Puma, anti-Bim, anti-phospho Mcl-1, anti-Mcl-1(WB), anti-cleaved caspase-3, anti-cleaved caspase-8, anti-cleaved caspase-9, anti-phospho STAT3, anti-STAT3, and anti-PARP-1 were purchased from Cell Signaling (Beverly, MA, USA). Anti-actin antibody was purchased from Sigma (Sigma, St. Louis, MO). Anti-Mule antibody was purchased from Abcam (Cat. No. ab70161). For the secondary antibodies, anti-mouse-IgG-HRP and anti-rabbit-IgG-HRP were purchased from Cell Signaling (Beverly, MA, USA).

Park S.H., Lee D.H., Kim J.L., Kim B.R., Na Y.J., Jo M.J., Jeong Y.A., Lee S.Y., Lee S.I., Lee Y.Y, & Oh S.C. (2016). Metformin enhances TRAIL-induced apoptosis by Mcl-1 degradation via Mule in colorectal cancer cells. Oncotarget, 7(37), 59503-59518.

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Western Blot Analysis of Signaling Proteins

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Cells were lyzed with RadioImmunoPrecipitation Assay (RIPA) buffer (Thermo Fisher Scientific, Villebon sur Yvette, France). Cell lysates were clarified by centrifugation at 14000 × g at 4°C for 15 min. For western blotting, proteins were separated by SDS-PAGE gels and transferred to a nitrocellulose membrane. Then, membranes were blocked with Tris buffered saline (TBS) (0.02 M Tris-HCl, 0.137 M NaCl, pH 7.4) containing 0.1% Tween (TBS-T) and 5% non-fat dry milk at room temperature during 1 hour and incubated overnight at 4°C with the following primary antibodies: anti-phospho-JAK2 (Santa Cruz Biotechnology), anti-GAPDH, anti-DDR1, anti-phospho-SHP2, anti-SHP2, anti-JAK2, anti-phospho-ERK1/2, anti-ERK1/2, anti-p21^CIP1 (Cell signaling Technology, Saint Quentin Yvelines, France), anti-DDR2 (R&D systems, Lille, France), anti-RAGE (GeneTex, Irvine, CA). Membranes were washed with TBS-T and incubated with the corresponding peroxidase conjugated secondary antibody at room temperature for 1 hour. Chemiluminescent detection was realized by using an ECL Prime Kit (GE Healthcare, Orsay, France).

Saby C., Buache E., Brassart-Pasco S., El Btaouri H., Courageot M.P., Van Gulick L., Garnotel R., Jeannesson P, & Morjani H. (2016). Type I collagen aging impairs discoidin domain receptor 2-mediated tumor cell growth suppression. Oncotarget, 7(18), 24908-24927.

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Western Blot Analysis of Apoptosis and Signaling

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Cell pellets were lysed in RIPA buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 1 mM DTT, 1 mM NaF, 1 mM sodium vanadate, 1 mM PMSF (Sigma), and 1% protease inhibitors cocktail (Merck). Protein extracts were quantitated and loaded on 8% to 12% sodium dodecyl sulfate polyacrylamide gel, electrophoresed, and transferred onto a PVDF membrane (Millipore). The membrane was incubated with primary antibody, washed, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce). Detection was performed by using a chemiluminescent Western detection kit (Cell Signaling Technology). The antibodies used were Anti-caspase-3, Anti-caspase-9, Anti-PARP, Anti-pSTAT3 (Y705), Anti-Jak2, Anti-pJak2 (Y1007/Y1008), Anti-pSTAT3 (S727), Anti-STAT1, Anti-pSTAT1 (Y701), Anti-STAT5, and Anti-pSTAT5 (Y694) (Cell Signaling Technology), Anti-Lamin B, Anti-Cyclin D1 (Santa Cruz Biotechnology), Anti-Mcl-1, Anti-STAT3 (Proteintech), and Anti-GAPDH (Abmart) antibodies.

Feng T., Cao W., Shen W., Zhang L., Gu X., Guo Y., Tsai H.I., Liu X., Li J., Zhang J., Li S., Wu F, & Liu Y. (2016). Arctigenin inhibits STAT3 and exhibits anticancer potential in human triple-negative breast cancer therapy. Oncotarget, 8(1), 329-344.

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Leelamine Modulates CXCR7/CXCR4 Signaling

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Leelamine (LEE, Figure 1A) was purchased from Cayman Chemical (Ann Arbor, MI, USA). LEE stock solution (10 mM) was prepared in EtOH, storage at −20 °C and finally diluted in cell culture medium for use. Anti-CXCR7 and anti-CXCR4 antibodies were purchased from abcam (Cambridge, UK). Anti-MnSOD, anti-fibronectin, anti-vimentin, anti-MMP-9, anti-MMP-2, anti-N-cadherin, anti-E-cadherin, anti-twist, anti-snail, anti-occludin, and anti-b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-STAT3(Tyr705), anti-STAT3, anti-phospho-JAK1(Tyr1022/1023), anti-JAK1, anti-phospho-JAK2(Tyr1007/1008), and anti-JAK2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Alexa Fluor^® 488 donkey anti-rabbit IgG (H+L) antibody and Alexa Fluor^® 594 donkey anti-mouse IgG (H+L) antibody was obtained from Life Technologies (Grand Island, NY, USA). The GSH/GSSG-Glo™ Assay kit was purchased from Promega (Madison, WI, USA).

Jung Y.Y., Um J.Y., Sethi G, & Ahn K.S. (2022). Potential Application of Leelamine as a Novel Regulator of Chemokine-Induced Epithelial-to-Mesenchymal Transition in Breast Cancer Cells. International Journal of Molecular Sciences, 23(17), 9848.

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Breast Cancer Protein Expression Analysis

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Proteins isolated from breast cancer cells and mammospheres were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Burlington, MA, USA). The blots were blocked in 5% skim milk in 1X PBS-Tween 20 at room temperature for 60 min and then incubated overnight at 4 °C with primary antibodies. The antibodies were anti-JAK2, anti-Stat3, anti-p65, anti-pp65, anti-lamin B, anti-phospho-Stat3 (Cell Signaling, Danvers, MA, USA), and anti-β-actin (Santa Cruz Biotechnology, Dallas, TA, USA) antibodies. After washing, the blots were detected with IRDye 680 RD and 800 CW secondary antibodies, and images were detected by using ODYSSEY CLx (LI-COR, Lincoln, NE, USA).

Choi H.S., Kim J.H., Kim S.L, & Lee D.S. (2019). Disruption of the NF-κB/IL-8 Signaling Axis by Sulconazole Inhibits Human Breast Cancer Stem Cell Formation. Cells, 8(9), 1007.

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Profiling Kinase Signaling in K562 Cells

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Proteins were extracted from K562 cells cultured with DMSO, 1.0 μM daphnoretin, 0.5 μM midostaurin (a PKC inhibitor), or TPA at 0.1 μM for 24, 48, and 72 h using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma Aldrich, St. Louis, MI, USA) with protease and phosphatase inhibitors (Roche, Indianapolis, IN, USA). Western blotting was performed according to a previous study [32 (link)]. Membranes were incubated overnight at 4 °C with the following primary antibodies at a 1:1000 dilution: anti-JAK2, anti-phospho-JAK2 (Tyr1007/1008), anti-STAT5, anti-phospho-STAT5 (Tyr694), anti-phospho-STAT3 (Tyr705) antibody (all from Cell Signaling Technology, MA, USA), and anti-STAT3 antibody (BD Biosciences, San Jose, CA, USA). Protein expression was detected using the ChemiGenius Bio Imaging System (Syngene, Frederick, MD, USA). The intensity of the bands was quantified using ImageJ software and levels were normalized to those of the internal control, β-actin.

Huang Y.C., Huang C.P., Lin C.P., Yang K.C., Lei Y.J., Wang H.P., Kuo Y.H, & Chen Y.J. (2022). Naturally Occurring Bicoumarin Compound Daphnoretin Inhibits Growth and Induces Megakaryocytic Differentiation in Human Chronic Myeloid Leukemia Cells. Cells, 11(20), 3252.

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