Phospho frs2

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-FRS2 is a lab equipment product designed to detect and quantify the phosphorylation of the FRS2 protein. FRS2 is an adaptor protein that plays a crucial role in various cellular signaling pathways. The Phospho-FRS2 product enables researchers to investigate the activation and regulation of FRS2 signaling in their research applications.

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6 protocols using phospho frs2

Antibody Profiling for Signaling Pathways

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The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): Phospho‐KIT (Tyr703; D12E12) rabbit mAb (no. 3073), Phospho‐KIT (Tyr719) antibody (no. 3391), Phospho‐anti‐KIT (pY823) rabbit mAb (no. 77522), KIT (Ab81) mouse mAb (no. 3308), Phospho‐AKT (Thr308; 244F9) rabbit mAb (no. 4056), Phospho‐AKT (Ser473; D9E) XP rabbit mAb (no. 4060), AKT (pan; C67E7) rabbit mAb (no. 4691), Phosphop44/42 MAPK (ERK1/2; Thr202/Tyr204; 197G2) rabbit mAb (no. 4377), p44/42 MAPK(ERK1/2; 137F5) rabbit mAb (no. 4695), PhosphoSTAT3 (Tyr705; D3A7) XP rabbit mAb (Biotinylated; no. 4093), STAT3 (D3Z2G) rabbit mAb (no. 12640), GAPDH (D16H11) XP rabbit mAb (no. 5174), Phospho‐FRS2 (Tyr196) antibody (no. 3864), Phospho‐Histone H2A.X (Ser139; 20E3) rabbit mAb (no. 9718), PARP (46D11) rabbit mAb (no. 9532), and caspase‐3 (8G10) rabbit mAb (no. 9665). β‐actin antibody was purchased from TransGen Biotech (Beijing, China; no. HC201‐02).

Liu J., Gao J., Wang A., Jiang Z., Qi S., Qi Z., Liu F., Yu K., Cao J., Chen C., Hu C., Wu H., Wang L., Wang W., Liu Q, & Liu J. (2022). Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours. Molecular Oncology, 16(8), 1761-1774.

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Protein Expression Analysis by Western Blot

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Total proteins were extracted from cells using the CHAPS lysis buffer (Beyotime, China). The quantification of protein was measured by the Bradford assay kit (Sigma). Equal amount of protein (10 μg) was loaded onto 10% SDS-PAGE and transferred to PVDF membrane (Millipore). The membrane was blocked with 5% skimmed milk for 1 h at room temperature. Then, the membrane was incubated with primary antibody at 4°C overnight. The following antibodies were used: phospho-ERK (cat: 4370; dilution: 1 : 1000; Cell Signaling Technology), ERK (cat: 4696; dilution: 1 : 1000; Cell Signaling Technology), phospho-p38 (cat: 4511; dilution: 1 : 1000; Cell Signaling Technology), p38 (cat: 8690; dilution: 1 : 1000; Cell Signaling Technology), phospho-FRS2 (cat: 3864; dilution: 1 : 1000; Cell Signaling Technology), FRS2 (cat: ab183492; dilution: 1 : 1000; Abcam), NOD2 (cat: ab31488; dilution: 1 : 1000; Abcam), EZH2 (cat: ab191250; dilution: 1 : 1000; Abcam), H3K27me3 (cat: ab6002; dilution: 1 : 1000; Abcam), and GAPDH (cat: ab8245; dilution: 1 : 5000; Abcam). Secondary antibodies conjugated to horseradish peroxidase were ordered from Sigma-Aldrich (USA). The results were visualized using Tanon™ High-sig ECL Western Blotting Substrate (Tanon, China) in Tanon-4600 instrument (Tanon, China). All experiments were repeated at least three times.

Duan A., Li H., Yu W., Zhang Y, & Yin L. (2022). Long Noncoding RNA XIST Promotes Resistance to Lenvatinib in Hepatocellular Carcinoma Cells via Epigenetic Inhibition of NOD2. Journal of Oncology, 2022, 4537343.

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FGF2-Induced Signaling Pathway Analysis

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Cells were plated in DMEM/10% FBS supplemented with 2 ng/mL FGF2 and treated for 6 h with inhibitors or vehicle. Lysates were generated as previously described²⁸. For immunoblot analysis, the following antibodies were used: phospho-FRS2, phospho-p44/42 MAPK (Erk1/ 2), total p44/p42 MAPK (Erk½), and actin (Cell Signaling Technologies); FGFR1 antibody was obtained from Abcam; ERa antibody was obtained from SantaCruz. Immunoreactive proteins were visualized by enhanced chemiluminescence (Pierce).

Drago J., Formisano L., Juric D., Niemierko A., Servetto A., Wander S., Spring L., Vidula N., Peppercorn J., Younger J., Malvarosa G., Yuen M., Sgroi D., Isakoff S.J., Moy B., Ellisen L.W., Iafrate J., Arteaga C.L, & Bardia A. (2019). FGFR1 gene amplification mediates endocrine resistance but retains TORC sensitivity in metastatic hormone receptor positive (HR+) breast cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(21), 6443-6451.

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Western Blot Analysis of Protein Expression

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Protein was extracted with lysis buffer (Beyotime, Shanghai, China) and then quantified by BCA Protein Quantitation Kit (Beyotime, Shanghai, China). Equal amounts of protein (30 μg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MD) Later on. The membrane was blocked with 5% bovine serum albumin (BSA) and incubated with primary antibodies overnight at 4 °C. After incubation with secondary antibodies for 1 h at room temperature, the proteins were detected by enhanced chemiluminescence reagents. Primary antibodies against β-catenin (51067-2-AP), GAPDH (60004-1-Ig), and E-cadherin (20874-1-AP) were purchased from Proteintech Group. Antibodies against LHX2 (ab243030), Vimentin (ab8978), FGF1 (ab179455), ZEB1 (ab203829), FGFR1 (ab76464), FGFR2 (ab109372), FRS (ab183492) and TWIST1 (ab50581) were purchased from Abcam. Antibodies against STAT3 (#9139), Phospho-STAT3 (#9145), ERK (#4695), Phospho-ERK (#4370), AKT (#2920), Phospho-AKT (#4060), Phospho-GSK3 (#5558), Phospho-FGFR (#3471) and Phospho-FRS2 (#3861) were purchased from Cell Signalling Technology.

Xie T., Du K., Liu W., Liu C., Wang B., Tian Y., Li R., Huang X., Lin J., Jian H., Zhang J, & Yuan Y. (2022). LHX2 facilitates the progression of nasopharyngeal carcinoma via activation of the FGF1/FGFR axis. British Journal of Cancer, 127(7), 1239-1253.

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FGFR Signaling Pathway Activation

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Example 12

Cells were seeded on tissue culture plates for 24 hours, pre-treated with 10 μg/ml FGFR blocking or control anti-gD antibody, then stimulated with 25 ng/ml FGF-7 (R&D Systems) in the presence of 20 μg/ml heparin (Sigma) for 15 minutes. Cells were placed on ice and protein immediately harvested with IP lysis buffer (Thermo Scientific). Protein lysates were passed through a syringe, cleared by centrifugation, then quantified using BCA protein assay (Thermo Scientific). Protein was separated on 4-12% Bis-Tris gels (Life Technologies), transferred to nitrocellulose membranes, blocked with 5% BSA or milk in TBST for 30 minutes, then blotted with primary antibody overnight at 4 C. Antibodies used: phospho-FGFR (Y653/654), phospho-FRS2 (Y196), phospho-ERK1/2 (T202/Y204), ERK1/2, phospho-AKT (S473), AKT, phospho-HER3 (Y1289), HER3, phospho-PLCgammal (Y783), PLCgammal (Cell Signaling); FGFR2, FRS2 (Santa Cruz Biotechnology); beta-actin (Sigma). Membranes were washed and incubated with appropriate HRP conjugated secondary antibodies for 1 hour, then washed and detected with SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific). Luminescence signal was acquired with FluorChem Q (Alpha Innotech).

US10208120B2. Anti-FGFR2/3 antibodies and methods using same (2019-02-19). Genentech, Inc. [US]. Inventors: Yiyuan Yin [US], Avi Ashkenazi [US], Paul J. Carter [US], Mark Chen [US], Junichiro Sonoda [US].

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FGF2 and FGFR1 Signaling Pathway Assay

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Recombinant FGF2 was purchased from Gibco (#PHG0024). Heparin sulfate was purchased from StemCell Technologies (#07980). The following antibodies were used (all primary antibodies are rabbit derived unless otherwise noted): anti-FGFR1 (#9740), phospho-FGFR (Y653/654, #3476, mouse derived), phospho-FRS2 (Y436, #3861; Y196, #3864), PLCγ (#5690), phospho-PLCγ (Y783, #2821), ERK (#4695), phospho-ERK (T202/Y204 #4370), Akt (#4691), phospho-Akt (S473 #4060), STAT3 (#4904), phospho-STAT3 (Y705, #9145), E-cadherin (#3195), Vimentin (#5741), ZEB1 (#3396), N-cadherin (#13116), HIF1α (#14179), Bcl-XL (#2764), Hsp90 (#4877), FGF2 (#61977), TGFβ (#3711), Cyclin D1 (#2978), GAPDH (#2118), horseradish peroxidase (HRP)-linked rabbit IgG secondary antibody (#7074) and HRP-linked mouse IgG secondary antibody (#7076), from Cell Signaling Technology. Anti-FRS2 (#10425, mouse derived) was purchased from Abcam. AZD4547 was purchased from Selleck Chemistry. The following CyTOF antibodies were purchased from Fluidigm Inc.: STAT3 (#3173003A), pSTAT3 (#3158005A), Vimentin (#3154014A), TGFβ (#3163010B), SOX2 (#3150019B), and Nanog (#3169014A). Custom antibody-metal conjugations were performed using kits purchased from Fluidigm, Inc. (PRD002) and carrier-free antibodies targeting FGFR1 (#9740), pFGFR1 (#3476), pFRS2 Y196 (#3864), and ZEB1 (#3396) were purchased from Cell Signaling Technology.

Ryan M.R., Sohl C.D., Luo B, & Anderson K.S. (2018). The FGFR1 V561M Gatekeeper Mutation Drives AZD4547 Resistance through STAT3 Activation and EMT. Molecular cancer research : MCR, 17(2), 532-543.

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