Fgfr2

Manufactured by Santa Cruz Biotechnology

Sourced in United States

FGFR2 is a laboratory equipment product developed by Santa Cruz Biotechnology. It is a receptor tyrosine kinase that plays a role in the regulation of cell growth, differentiation, and angiogenesis.

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10 protocols using fgfr2

CEMP1 Protein Overexpression Validation

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To confirm the overexpression of CEMP1 at the protein level, total proteins were isolated from HGF/CEMP1, NIH-3T3/CEMP1, HGF and NIH-3T3 and western blots were performed using anti-human rhCEMP1 polyclonal antibodies [10 (link)]. Validation of selected genes was done using, anti-E-Cadherin, anti-N-Cadherin, anti-fibroblast growth factor receptor (FGFR-2), anti-high mobility group (HMG) proteins (HMG-1/2/3 (FL-215), anti-heparin binding epidermal-like growth factor (HB-EGF), anti-HoxA5 and anti-c-Met oncogene were used (Santa Cruz Biotech, CA, USA). Total proteins were isolated from cell layer cultures and all the antibodies were produced against human proteins.

Bermúdez M., Imaz-Rosshandler I., Rangel-Escareño C., Zeichner-David M., Arzate H, & Mercado-Celis G.E. (2015). CEMP1 Induces Transformation in Human Gingival Fibroblasts. PLoS ONE, 10(5), e0127286.

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Comprehensive Western Blot Protocol

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The protocol used for western blot has been previously described in detail [29 (link)]. In brief, cells were harvested, washed twice with PBS, and lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer. Lysates were boiled in 4× SDS-sample buffer and resolved on SDS-PAGE. SDS-PAGE-separated proteins were transferred by electrophoresis onto a nitrocellulose membrane. Finally, blots were probed with appropriate primary and appropriate secondary antibodies, as follows: p-Rb (9307; Cell Signaling Technology, Danvers, MA, USA), CCNE2 (11935-1-AP; Proteintech), p-ERK (SC-7383; Santa Cruz Biotechnology, Dallas, TX, USA), ERK (SC-154; Santa Cruz Biotechnology), FGFR2 (SC-6930; Santa Cruz Biotechnology), CDK2 (SC-6248; Santa Cruz Biotechnology), CDK4 (SC-23896; Santa Cruz Biotechnology), CDK6 (19117-l-AP; Proteintech), CDKN2A/p16 INK4a (ab16880; Abcam, Cambridge, UK) and β-actin (SC-47778; Santa Cruz Biotechnology). Densitometry readings/intensity ratio of each band. In addition, the whole blot showing all the bands with all molecular weight markers on the Western in the Supplementary Figure S5.

Lin C.Y., Wu R.C., Huang C.Y., Lai C.H., Chao A.S., Li H.P., Tsai C.L., Kuek E.J., Hsu C.L, & Chao A. (2021). A Patient-Derived Xenograft Model of Dedifferentiated Endometrial Carcinoma: A Proof-of-Concept Study for the Identification of New Molecularly Informed Treatment Approaches. Cancers, 13(23), 5962.

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Immunohistochemical Analysis of Parathyroid Tissues

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Paraffin-embedded paraformaldehyde-fixed thyro-parathyroid tissues were sectioned serially at 6-μm thickness. One in every six sections was stained with hematoxylin and eosin (HE) to determine the location of the parathyroid glands. Then, the sections containing the parathyroid glands were immunostained using the standard indirect immunofluorescence technique. The primary antibodies used in the study were for PTH (sc-9676; Santa Cruz Biotechnology, Dallas, TX), Ki67 (ab16667; Abcam, Cambridge, UK), phosphorylated ERK1/2 (#4370; Cell Signaling Technology, Danvers, MA), CaSR (MA1-934; Thermo Fisher Scientific K.K.), VDR (sc-1009; Santa Cruz Biotechnology), αKlotho (AF1819; R&D Systems), FGFR1 (sc-121; Santa Cruz Biotechnology), FGFR2 (sc-122; Santa Cruz Biotechnology), FGFR3 (sc-123; Santa Cruz Biotechnology), and FGFR4 (sc-9006; Santa Cruz Biotechnology) with DAPI (Thermo Fisher Scientific K.K.) co-staining for nuclei. The secondary
antibodies used for indirect immunofluorescence staining were as follows: Alexa Fluor 568 or 488-conjugated anti-rabbit, -mouse, or -goat IgG produced in goat or donkey (Molecular Probes).

Kawakami K., Takeshita A., Furushima K., Miyajima M., Hatamura I., Kuro-o M., Furuta Y, & Sakaguchi K. (2017). Persistent fibroblast growth factor 23 signalling in the parathyroid glands for secondary hyperparathyroidism in mice with chronic kidney disease. Scientific Reports, 7, 40534.

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Evaluating FGFR2 Expression in Cutaneous Squamous Cell Carcinoma

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An IRB approved retrospective study was performed to identify patients with cSCC at LSU Health-Shreveport and Overton Brooks Veterans Affairs Medical Center. Normal skin samples (n=9) were collected from Caucasian patients undergoing reduction mammoplasty surgery. Normal skin, Actinic Keratosis (n = 9) cSCC sections (n = 28) and cSCC with metastasis (n = 21) were immunostained for FGFR2 (1:600, Santa Cruz Biotechnology) using DAB as a chromogen, and the staining intensity was quantified by a registered pathologist who was blinded to the clinical details as described before (21). A semi-quantitative approach was used to score the staining intensity. Slides were categorized based on the following: no staining scored as (0), weak or focal staining scored as (1+), moderate staining scored as (2+), and strong staining scored as (3+). Please note that part of the data depicting FGFR2 expression in normal and cSCC is already published in our previous research manuscript ^{29 (link)}. However, for this research study we have increased the sample size and have now included the AK group as well.

Khandelwal A.R., Kent B., Hillary S., Alam M.M., Ma X., Gu X., DiGiovanni J, & Nathan C.A. (2019). Fibroblast growth factor receptor (FGFR) promotes progression of cutaneous squamous cell carcinoma (cSCC). Molecular carcinogenesis, 58(10), 1715-1725.

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Comprehensive Western Blot Analysis

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For western blot analysis, samples were isolated from the lysate (30 μg), mixed in reducing buffer, boiled, resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane by blotting. The blot was incubated overnight at 4°C in a blocking solution with primary antibodies to the following antigens: α-amylase, AQP5, ZO-1, CK7, CK18, FGF7, FGFR2, p53 upregulated modulator of apoptosis (PUMA) and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA); E-cadherin (BD Pharmingen, San Diego, CA, USA); p53, Bax (Abcam, England); Bcl-2, Cytochrome C, Cleaved caspase-9 and Cleaved caspase-3, protein kinase B (PI3K), p-PI3K, Akt, p-Akt and murine double minute 2 (MDM2), p-MDM2 (Cell signaling, Danvers, MA, USA). After washing the blots with 0.1% Tween 20 in 1×PBS, they were incubated with horseradish peroxidase-conjugated secondary antibodies corresponding to each primary antibody, after which they were subjected to enhanced chemiluminescence detection (GE Healthcare Life Science, USA).

Choi J.S., Shin H.S., An H.Y., Kim Y.M, & Lim J.Y. (2017). Radioprotective effects of Keratinocyte Growth Factor-1 against irradiation-induced salivary gland hypofunction. Oncotarget, 8(8), 13496-13508.

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Comprehensive Protein Expression Analysis

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Abcam: EpCAM. Bethyl Laboratories: BRD4. Cell Signaling Technology: AKT, BIM, CDK9, DDR1, IGF1R, KDR, KIT, MEK1/2, MYC, pAKT (S473), pAKT (T308), PDGFRB, pERK1/2 (T202,Y204), pSRC (Y416), vimentin. Santa Cruz Biotechnology: ERK2, FGFR2.

Zawistowski J.S., Bevill S.M., Goulet D.R., Stuhlmiller T.J., Beltran A.S., Olivares-Quintero J.F., Singh D., Sciaky N., Parker J.S., Rashid N.U., Chen X., Duncan J.S., Whittle M.C., Angus S.P., Velarde S.H., Golitz B.T., He X., Santos C., Darr D.B., Gallagher K., Graves L.M., Perou C.M., Carey L.A., Earp H.S, & Johnson G.L. (2017). Enhancer Remodeling During Adaptive Bypass to MEK Inhibition Is Attenuated by Pharmacological Targeting of the P-TEFb Complex. Cancer discovery, 7(3), 302-321.

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Western Blot Analysis of FGFR and Downstream Signaling

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Cells were lysed using PRO-PRE Protein Extraction Solution (Intron Biotechnology, Seongnam, Korea), and protein concentration was determined using a Bradford assay kit (BIO-RAD, Hercules, CA, USA). Cell lysates (40 µg) were separated on 8% or 10% acrylamide gels by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Hybond-ECL nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK). Membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature. The blots were probed using primary antibodies against FGFR1, FGFR2 (Santa Cruz Biotechnology, USA), FGFR3, FGFR4 (Abcam), phospho-FGFR1 (p-FGFR1), total-c-Met, phospho-c-Met (p-c-Met), total-AKT, phospho-AKT (p-AKT), total-ERK, phospho-ERK (p-ERK) (Cell Signaling Technology), or β-actin (Santa Cruz Biotechnology). Membranes were then incubated with horseradish peroxidase–conjugated anti-rabbit or anti-mouse secondary antibodies (GE Healthcare, Piscataway, NJ, USA). Bands were visualized using an enhanced chemiluminescence (ECL) kit (Amersham Biosciences, Buckinghamshire, UK) following the manufacturer’s protocols.

Lee Y.Y., Ryu J.Y., Cho Y.J., Choi J.Y., Choi J.J., Choi C.H., Sa J.K., Hwang J.R, & Lee J.W. (2024). The anti-tumor effects of AZD4547 on ovarian cancer cells: differential responses based on c-Met and FGF19/FGFR4 expression. Cancer Cell International, 24, 43.

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Cell Cycle Regulation Protein Analysis

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Preparation of cell lysate was achieved by solubilization in RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NaDeoxycholate, 1% Triton-X 100, 0.1% SDS) containing protease inhibitor cocktail (Cell Signaling), followed by removal of insoluble material by centrifugation. Samples were separated by SDS polyacrylamide gel electrophoresis and transferred onto Immobilon. Immunoreactive protein bands were visualized by incubation with primary and HRP conjugated secondary antibodies (Promega, W402B; Sigma-Aldrich, A0545), and enhanced chemiluminescence (Thermo Fisher). The primary antibodies utilized include total and/or phospho-specific antibodies for Cyclin E1 (Cell Signaling, 20808), Cyclin A2 (Abcam, ab38), Cyclin B1 (Cell Signaling, 4138), Cyclin D1 (Cell Signaling, 2978), CDK1 (Cell Signaling, 77055; Cell Signaling, 4539), CDK2 (Cell Signaling, 2546), Aurora B (Cell Signaling, 3094; Cell Signaling, 2914), Histone H3 (Cell Signaling, 9715; Cell Signaling, 3377), Erk1/2 (Cell Signaling, 4695; Cell Signaling, 9101), FGFR2 (Santa Cruz, sc-122) and βtubulin (Cell Signaling, 5346).

Nam H.K., Vesela I., Siismets E, & Hatch N.E. (2018). Tissue Nonspecific Alkaline Phosphatase Promotes Calvarial Progenitor Cell Cycle Progression and Cytokinesis via Erk1,2.. Bone, 120, 125-136.

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FGF-Mediated FGFR Activation Dynamics

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Cells exposed to NVP‐BGJ398 for 3 h and either 25 ng·mL⁻¹ FGF7 or 150 ng·mL⁻¹ FGF19 for 20 min were harvested in 1xRIPA containing Complete mini protease inhibitor (Roche Cat#4693159001) and Phosphatase Inhibitor cocktails (Sigma Aldrich Cat#P0044 and P5726). 1.2 mg of protein lysate was precleared with 1.5 mg Protein A Dynabeads (Thermo Scientific Cat#10001D) on a rotator for 2 h at 4 °C before pulldown with 4 μg of either FGFR2 (Santa Cruz Biotechnologies, Cat#sc‐122, RRID:AB_631509), FGFR4 (Santa Cruz Biotechnologies, Cat#sc‐124, RRID:AB_631512), or normal rabbit IgG (Cell Signaling Technologies, Cat#2729, RRID:AB_1031062) as described previously [30 ].

Milton C.I., Selfe J., Aladowicz E., Man S.Y., Bernauer C., Missiaglia E., Walters Z.S., Gatz S.A., Kelsey A., Generali M., Box G., Valenti M., de Haven‐Brandon A., Galiwango D., Hayes A., Clarke M., Izquierdo E., Gonzalez De Castro D., Raynaud F.I., Kirkin V, & Shipley J.M. (2021). FGF7–FGFR2 autocrine signaling increases growth and chemoresistance of fusion‐positive rhabdomyosarcomas. Molecular Oncology, 16(6), 1272-1289.

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Immunoblotting with Cellular Markers

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Golgin 97 and Pan-Cadherin from Cell Signalling Technology. Calreticulin from Genetex. Py99, FGFR2, Flotillin-1, GRB2 antibodies from Santa Cruz. c-CBL from ThermoScientific. Lipid standards were from Larodan, Sweden. Solutes for HPTLC were from Sigma.

Rohwedder A., Knipp S., Roberts L.D, & Ladbury J.E. (2021). Composition of receptor tyrosine kinase-mediated lipid micro-domains controlled by adaptor protein interaction. Scientific Reports, 11, 6160.

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