Phospho alky1604

Manufactured by Cell Signaling Technology

Phospho-ALKY1604 is a laboratory reagent produced by Cell Signaling Technology. It is designed to detect and quantify the phosphorylation of the ALKY1604 protein target using immunodetection techniques.

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Lab products found in correlation

6 protocols using phospho alky1604

Antibody-based Analysis of ALK Signaling

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Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA). Pan-ERK1/2 (1:2000; 610123), was purchased from BD Transduction Laboratories (Franklin Lakes, NJ). Ki67 was from BD Pharmingen, (1:100; 558616). Monoclonal antibody 135 (anti-ALK) was described (Chand et al., 2013 (link); Moog-Lutz et al., 2005 (link)). Horseradish-peroxidase-conjugated secondary antibodies goat anti-rabbit IgG (PIEA31460) and goat anti-mouse IgG (PIEA31394; both at 1:5000) were from Thermo Scientific (Waltham, MA).
Crizotinib was from Haoyuan Chemexpress Co., Limited, Shanghai, China. PF-04643922 was from Pfizer, California, USA.

Guan J., Tucker E.R., Wan H., Chand D., Danielson L.S., Ruuth K., El Wakil A., Witek B., Jamin Y., Umapathy G., Robinson S.P., Johnson T.W., Smeal T., Martinsson T., Chesler L., Palmer R.H, & Hallberg B. (2016). The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN. Disease Models & Mechanisms, 9(9), 941-952.

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IHC Evaluation of Protein Expression

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Protein expression was detected by IHC in serial sections of formalin-fixed paraffin-embedded tumour samples according to the manufacturer's instructions for each antibody. The samples were stained with antibodies against total EGFR, phospho-EGFR (Y1068), total ALK, and phospho-ALK (Y1604) (Cell Signalling Technology), followed by incubation in rabbit monoclonal anti-human ALK antibody (#3633 WP1-01; clone D5F3) at a dilution of 1:100. The staining intensity was scored from 0 to 3+; tumours with no expression (0) were described as negative for ALK protein expression, while tumours scored as 1+, 2+, or 3+ were deemed positive [45 (link), 46 (link)], and the staining intensity was scored according to the following criteria: 3+, intense, granular cytoplasmic staining; 2+, moderate, smooth cytoplasmic staining; 1+, faint cytoplasmic staining in 10% of tumor cells; and 0, no staining [45 (link)], and high phosphorylation of proteins means IHC ++ or +++; low phosphorylation of proteins means IHC + or +/− NA, not available. -/NA, IHC negative because of not available testing [20 (link), 38 ].

Lou N.N., Zhang X.C., Chen H.J., Zhou Q., Yan L.X., Xie Z., Su J., Chen Z.H., Tu H.Y., Yan H.H., Wang Z., Xu C.R., Jiang B.Y., Wang B.C., Bai X.Y., Zhong W.Z., Wu Y.L, & Yang J.J. (2016). Clinical outcomes of advanced non-small-cell lung cancer patients with EGFR mutation, ALK rearrangement and EGFR/ALK co-alterations. Oncotarget, 7(40), 65185-65195.

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Evaluating Therapeutic Responses in Oncology

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Antibodies against histone H2A.X (H2AX), γ-H2AX (S139), ATM, phospho-ATM (S1981), cyclin D1, cleaved PARP, caspase-3, p53, phospho-p53 (S15), acetyl-p53 (K382), p44/42 mitogen-activated protein kinase (MAPK), phospho-p44/42 MAPK (T202/Y204), AKT, phospho-AKT (S473), histone 2B (H2B), acetyl-H2B (K5), histone deacetylase 1 (HDAC1), O-6-methylguanine-DNA methyltransferase (MGMT), signal transducer and activator of transcription 3 (STAT3), phospho-STAT3 (Y705), anaplastic lymphoma kinase (ALK), phospho-ALK (Y1604), insulin-like growth factor receptor (IGF-1R), phospho-IGF-1R (Y1132), phospho-Src kinase (Y416) and GAPDH were purchased from Cell Signaling (Danvers, MA). SN-38 (S), Temozolomide (T), Vorinostat (V), MK-2206 and AZD3463 were from Selleck Chemicals (Houston, TX). Stock solutions of compounds were prepared in DMSO and stored at -20°C or diluted in media for experiments.

Sampson V.B., Vetter N.S., Kamara D.F., Collier A.B., Gresh R.C, & Kolb E.A. (2015). Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways. PLoS ONE, 10(11), e0142704.

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Immunoblotting Analysis of Metabolic Regulators

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Lysates from cells or xenografts were prepared in RIPA buffer supplemented with protease/phosphatase inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Proteins were analyzed by immunoblotting as previously described.^{58 (link)} The antibodies for ALK, β-tubulin, phospho-ALK^Y1604, HK1, HK2, PFK1, GAPDH, PKM2, LDHA, PGAM1, Enolase-1, AKT, phospho-AKT^S473 and phospho-4E-BP1^T37/46 were purchased from Cell Signaling. The antibodies for GLUT1 and GLUT3 were obtained from Abcam (Cambridge, MA). The HIF1α antibody was from BD Biosciences (San Jose, CA).

Ma Y., Yu C., Mohamed E.M., Shao H., Wang L., Sundaresan G., Zweit J., Idowu M, & Fang X. (2016). A causal link from ALK to hexokinase II overexpression and hyperactive glycolysis in EML4-ALK-positive lung cancer. Oncogene, 35(47), 6132-6142.

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Immunoblotting Analysis of Metabolic Regulators

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Molecular Signaling Pathway Analysis

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Ba/F3 cells and patient derived cell lines were treated for 6 hours with lorlatinib, LA7 or LA9 unless otherwise noted. Total protein lysates were analyzed by western blotting with the following antibodies and dilutions. (all from Cell Signaling Technology): phospho-ALK Y1604 (3341, 1:1000), phospho-ALK Y1282/83 (9687, 1:1000), ALK (3633, 1:1000), phospho-AKT S473 (4060, 1:1000), AKT (4691, 1:1000), phospho-ERK1/2 T202/Y204 (9101, 1:1000), ERK1/2 (9102, 1:1000), phospho-S6 S240/244 (5364, 1:10,000), S6 (2217, 1:10,000), and β-Actin (4970, 1:1000).

Shiba-Ishii A., Johnson T.W., Dagogo-Jack I., Mino-Kenudson M., Johnson T.R., Wei P., Weinrich S.L., McTigue M.A., Walcott M.A., Nguyen-Phuong L., Dionne K., Acker A., Kiedrowski L.A., Do A., Peterson J.L., Barth J.L., Yeap B.Y., Gainor J.F., Lin J.J., Yoda S, & Hata A.N. (2022). Analysis of lorlatinib analogs reveals a roadmap for targeting diverse compound resistance mutations in ALK-positive lung cancer. Nature cancer, 3(6), 710-722.

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