The cells were homogenized in lysis buffer (50mM Tris/HCl pH 7.4, 500mM NaCl, 1% NP-40, 0.5% Na-DOC, 0.1% SDS) supplemented with 20μg/ml complete protease inhibitor cocktail (Roche, Mannheim, Germany), 5mM NaF and 100μM Na-vanadate. Aliquots containing 20μg of protein were analyzed by electrophoresis on 10% polyacrylamide gels and transferred to polyvinylidene-difluoride membranes. Proteins were identified using antibodies to phospho-FRS2α (Cell signalling, Boston, MA; #3861), phospho-FGFR (Cell Signalling; #3471) and FGFR3 (#sc-123, recognizes both FGFR3 splice variants; Santa Cruz Biotechnology, Inc., Dallas, TX); ERK1/2 (Upstate, Lake Placid, NY #06-182; 1:5000), phospho-ERK (Cell signalling, sampler kit: phospho-p44/42 MAP Kinase Thr202/Tyr204; 1:5000), GSK3β (Cell signalling #9315; 1:1000), phospho-GSK3β (Cell signalling #9323; 1:1000), S6 (#2212, Cell signalling, Boston, MA; 1:5000), phospho-S6 (Cell signalling #2215; 1:5000). Band intensity was quantified using ImageQuant software (GE Healthcare).
Phospho fgfr
Manufactured by Cell Signaling Technology
Sourced in United States
The Phospho-FGFR product is a laboratory reagent used to detect and quantify the phosphorylation of fibroblast growth factor receptors (FGFRs) in biological samples. It provides a direct and specific method for measuring FGFR activation, which is a key signaling event in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Phospho akt, by Cell Signaling Technology (4 mentions)
Phospho erk, by Cell Signaling Technology (3 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Phospho erk1 2, by Cell Signaling Technology (2 mentions)
Phospho stat3, by Cell Signaling Technology (2 mentions)
Phospho frs2, by Cell Signaling Technology (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Phospho gsk3β, by Cell Signaling Technology (1 mentions)
Imagequant software, by GE Healthcare (1 mentions)
Phospho frs2 α, by Cell Signaling Technology (1 mentions)
Sc 123, by Santa Cruz Biotechnology (1 mentions)
Phospho s6, by Cell Signaling Technology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Phospho src tyr416, by Cell Signaling Technology (1 mentions)
Phospho akt thr308, by Cell Signaling Technology (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Vegfr2, by Cell Signaling Technology (1 mentions)
Femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
8 protocols using phospho fgfr
1
Western Blot Analysis of FGF Signaling
2
Protein Expression Analysis of Tumor Samples
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Total protein lysates from tumors were extracted by RIPA lysis buffer supplemented with proteinase and phosphatase inhibitors (Roche Diagnostics, 4693159001). The samples were separated on a 10% SDS‒PAGE gel, transferred to a nitrocellulose membrane (Millipore), and probed with the following antibodies: Fgfr2 (1:1000, Merck, sc-6930), phospho-Fgfr (1:1000, Cell Signaling Technology, 3476), Vegfr2 (1:1000, Cell Signaling Technology, 12599), phospho-Src (Tyr416) (1:1000, Cell Signaling Technology, 2101), β-actin (1:1000, Cell Signaling Technology, 3700), phospho-AKT (Ser473) (1:1000, Cell Signaling Technology, 9271), phospho-AKT (Thr308) (1:1000, Cell Signaling Technology, 13038), and EGFR (1:1000, Cell Signaling Technology, 8339). Full and uncropped western blots are presented in the Supplemental File.
3
Immunoblotting of FGF Receptor Signaling
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Total protein lysates were extracted from untreated cell lines as well as 48 h after siRNA transfection or 2 or 5 h after inhibitor treatment. For western blot analysis equal amounts of total protein were loaded on NuPAGE® Bis-Tris Gels (Life Technologies). Electrophoretically separated proteins were transferred to nitrocellulose membrane and unspecific binding was blocked with 5% milk/PBS-Tween. Membranes were incubated with primary antibodies overnight at 4°C (FGFR2 1:500; ACTB 1:5000, AC-15, both Sigma Aldrich; FGFR1 1:500; FGFR4 1:500, AM11076PU-N, both from Acris Antibodies, San Diego CA, U.S.A.; FGFR3 1:500, D2G7E; phospho-FGFR 1:500, 55H2; ERK1/2 1:1000; phospho-ERK1/2 1:1000, 20G11, all from CellSignaling Technology, Danvers MA, U.S.A.; HPRT 1:1000, abcam). After incubation with HRP-conjugated secondary antibody SuperSignal West Pico or Femto Chemiluminescent Substrate (Thermo Scientific) was used for detection.
4
Immunoblotting Analysis of Signaling Pathways
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Cell and tumor lysates were prepared in lysis buffer as described before [17 (link)]. Total protein amount was measured using the Bradford method (Bio-Rad, Hercules, CA, USA). 10–30 μg of proteins was subjected to electrophoresis on NuPage gels (Invitrogen). Immunoblotting was performed with antibodies recognizing phospho-FGFR (1:1000), AKT (1:1000), phospho-AKT (1:500), phospho-ERK (1:500), PARP (1:1000), cleaved PARP (1:1000), or β-actin (1:1000) from Cell Signaling Technology (Beverly, MA) and FGFR2 (1:400) (R&D, Minneapolis, MN, USA) or ERK2 (1:1000) (Santa Cruz Biotechnology, Dallas, TX, USA), followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:3000) (Cell Signaling; GE Healthcare, Little Chalfont, UK).
5
Comprehensive Signaling Pathway Assay
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Assays were performed as a previous study [44 (link)]. Antibodies were from Cell Signaling (Danvers, MA), including Phospho-FGFR (Tyr653/654) (#3471), Phospho-MEK1/2 (#9121), Phospho-p44/42 MAPK (#9106), p44/42 MAPK (#4695), Cyclin D1 (#2978), p21 (#2946), p27 (#2552), Phospho-Rb (Ser807/811) (#9308), cleaved PARP (Asp214) (#9541), PARP (#9542), LATS1 (#9153), LATS2 (#5888) Phospho c-Jun (Ser73) (#3270), c-Jun (#9165), c-Myc (#9402); Abcam (MA, US), including anti-FGFR2 (ab58201), and anti-YAP1 (ab52771); Immunoway (TX, US), including Bcl2 (YM3041) and β-Actin (YM3115); Santa Cruz (TX, US) including CTGF (sc-14939) and TEAD4 (TEF-3, sc-101184). The other antibodies include Phospho-LATS1/2 (Ser909/872) (AP0904, Abclonal), N-cadherin (#33-3900, ZYMED).
6
Quantifying Phosphorylation Signaling Cascades
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To reduce basal phosphorylation, cells were cultured for 24 h in growth factor/serum-free medium. Starved cells were pretreated with vehicle or sorafenib for different times (15–180 min), and then either EGF (20 ng/ml) or bFGF (10 ng/ml) was added for 10 min. Total protein content was measured from whole cell lysates as described previously [32 (link)]. Equal amounts of proteins were size-fractionated by SDS-PAGE, and transferred onto PVDF membrane (Bio-Rad Laboratories). Blots were probed with the following antibodies: phospho-ERK1/2 (Thr201-Tyr204, #9101), phospho-MEK (Ser217/221, #9121), phospho-Akt (Ser473, #9271) phospho-STAT3 (Ser-727, #9134), Mcl-1 (#5453), phospho-FGFR (Tyr653/654, #3476), and FGFR1 (#9740) from Cell Signaling and α-tubulin (#T5168; Sigma-Aldrich). Secondary mouse and rabbit HRP-linked antibodies were from GE Healthcare. Detection of immunocomplexes was performed using Clarity ECL Western Blot (Bio-Rad Laboratories). All experiments were repeated at least three times. Densitometric analysis of bands was performed using ChemiDoc imaging system (Bio-Rad Laboratories).
7
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).
8
FGF2 and FGFR1 Signaling Pathway Assay
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