Fgfr4

Manufactured by Abcam

Sourced in United States

FGFR4 is a receptor tyrosine kinase that plays a role in fibroblast growth factor signaling. It is a member of the fibroblast growth factor receptor family.

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Lab products found in correlation

Faststart universal sybr green master mix with rox, by Roche (1 mentions) Quantitech reverse transcription kit, by Qiagen (1 mentions) Sirt1, by Cell Signaling Technology (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Nf κb, by Cell Signaling Technology (1 mentions) Mini protean tetra cell system, by Bio-Rad (1 mentions) Rneasy kit, by Qiagen (1 mentions) Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions) Actin, by Merck Group (1 mentions) Fgfr3, by Santa Cruz Biotechnology (1 mentions) Fgfr1, by Santa Cruz Biotechnology (1 mentions) Phospho enos ser1177 p enos, by Cell Signaling Technology (1 mentions) Phosphatase inhibitor, by Sangon (1 mentions) P mtor, by Affinity Biosciences (1 mentions) P akt, by Affinity Biosciences (1 mentions) P fgfr3, by Abcam (1 mentions) Fgfr2, by Abcam (1 mentions) Fgfr3, by Abcam (1 mentions) Vegfr2, by Cell Signaling Technology (1 mentions) Gapdh, by Proteintech (1 mentions)

8 protocols using fgfr4

Comprehensive mRNA and Protein Analysis

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Liver, adipose tissues, and ileal samples were collected for mRNA analysis. The ileal samples were taken approximately 2 cm away from the ileo-cecal junction. Total RNA was extracted using the RNeasy kit (Qiagen) and cDNA was prepared using the Quantitech Reverse Transcription kit (Qiagen). Real-time quantitative PCR (qPCR) amplification was performed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories) with FastStart Universal SYBR Green Master Mix with ROX (Roche Diagnostics)9 (link)14 (link). Relative mRNA levels were calculated using the comparative cycle threshold (Ct) method and were normalized to the values of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels. The qPCR primers used in this study are listed in Table S1 (Supplementary Table S1).
Western blot analyses were performed by separating liver extracts by electrophoresis in 10% and/or AnyKd Mini-Protean TGX gels using Bio-Rad Mini-PROTEAN Tetra Cell System and transferred to PVDF membranes via Bio-Rad Blotting Module9 (link)14 (link). Multiple protein band quantification and analysis were performed using AlphaView software version 3.4 (ProteinSimple). SIRT1, NF-κB, acetylated NF-κB and GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA). Lcn2, Saa1, and FGFR4 antibodies were purchased from Abcam (Cambridge, MA).

Wang J., Kim C., Jogasuria A., Han Y., Hu X., Wu J., Shen H., Chrast R., Finck B.N, & You M. (2016). Myeloid Cell-Specific Lipin-1 Deficiency Stimulates Endocrine Adiponectin-FGF15 Axis and Ameliorates Ethanol-Induced Liver Injury in Mice. Scientific Reports, 6, 34117.

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Western Blot Analysis of Protein Targets

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Western blot technique was used for protein analysis of cell lysates as previously described [12 (link)]. Briefly, sample media was mixed with 4X loading (sample) buffer (Sigma, St Louis, MO) and Radio-Immuno Precipitation Assay buffer, pH 7.4 (Cat No. BP-115, Boston BioProducts, Ashland, MA). 15–50 μg of sample was subjected to Western blot depending on target proteins. The same amount of protein was used within a Western blot and target proteins were then visualized with an enhanced chemiluminescence detection system. Antibodies used in this study were for endothelial nitric oxide synthase (eNOS) (Cat No. 9572; Cell signaling technology, Danvers, MA) 1:1000; phospho-eNOS (Ser1177) (p-eNOS) (Cat No. 9571; Cell signaling technology, Danvers, MA) 1:1000; α-Klotho (Cat No. Ab75023; Abcam, Cambridge, MA) 1:1000; phospho-FGFR (Tyr653/654) (p-FGFR) (Cat No.3471; Cell signaling technology, Danvers, MA) 1:1000; FGFR1 (Cat No. SC-121; Santa Cruz Biotechnology, Santa Cruz, CA) 1:1000; FGFR3 (Cat No. SC-123; Santa Cruz Biotechnology, Santa Cruz, CA) 1:1000; FGFR4 (Cat No. Ab5481; Abcam, Cambridge, MA) 1:1000 and Actin (Cat No. MAB1501; Millipore, Billerica, MA) 1:1000.

Chung C.P., Chang Y.C., Ding Y., Lim K., Liu Q., Zhu L., Zhang W., Lu T.S., Molostvov G., Zehnder D, & Hsiao L.L. (2017). α-Klotho expression determines nitric oxide synthesis in response to FGF-23 in human aortic endothelial cells. PLoS ONE, 12(5), e0176817.

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Protein Expression Analysis by Western Blot

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Total protein was lysed with proteinase inhibitor cocktail and radioimmunoprecipitation assay (RIPA) buffer with and conditionally adding phosphatase inhibitors (Sangon Biotech, Shanghai, China). The protein samples were then mixed with 5 × loading buffer, then denatured at 95℃ for 5 min, and electrotransferred to polyvinylidene fluoride (PVDF) membrane by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After blocking with 5% skim milk, the membrane was incubated in the primary antibody overnight at 4 °C, and then the secondary antibody diluted in Tris-buffered saline with Tween 20 (TBST) was used. Then, the protein-antibody complexes were detected using the enhanced chemiluminescence (ECL) on the Tanon 5200 Multi intelligent imaging system. The primary antibody used in this study are shown as below: VEGFR1 (Affinity, AF6204), VEGFR2 (Cell signaling Technology, 9698), VEGFR3 (Affinity, AF4201), FGFR1 (Signalway Antibody, 49,175), FGFR2 (Abcam, ab109372), FGFR3 (Abcam, ab133644), FGFR4 (Abcam, ab119378), c-Kit (Bioworld Technology, BS2433), PDGFRβ (Bioworld Technology, BS1764), GAPDH (Proteintech, 60,004), p-FGFR3 (Abcam, ab155960), AKT (Affinity, AF6261), p-AKT (Affinity, AF0016), mTOR (Affinity, AF6308), p-Mtor (Affinity, AF3308), BCL2 (Proteintech, 12,789–1-AP), BAX (Abcam, ab32503), METTL3 (Proteintech, 15,073–1-AP).

Chen J., Li S., Huang Z., Cao C., Wang A, & He Q. (2022). METTL3 suppresses anlotinib sensitivity by regulating m6A modification of FGFR3 in oral squamous cell carcinoma. Cancer Cell International, 22, 295.

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Melanoma Cell Signaling Analysis

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Melanoma cells were lysed in RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4) supplemented with proteinase inhibitor cocktail. Then, 20–25 µg of proteins were separated by SDS-PAGE and Western blot analysis was carried out according to standard protocols using the following antibodies (obtained from Cell Signaling Technology (Danvers, MA, USA) and used at 1:1000 dilution unless otherwise mentioned): phosphoKIT (Tyr719), KIT (C-19; Santa Cruz Biotechnology, Dallas, TX, USA), phosphoSTAT3 (Tyr705), STAT3 (F-2; Santa Cruz, Dallas, TX, USA), phosphoAKT XP (ser473), AKT, phosphoERK, ERK phosphoP90RSK (Thr359/Ser363), P90RSK, BAD, BAK, BAX, BCL2, BCL-XL, BID, BIM, FGFR1, FGFR2 (C-8; Santa Cruz Biotechnology, Dallas, TX, USA), FGFR3 (B-9; Santa Cruz Biotechnology, Dallas, TX, USA), FGFR4, and Actin (Abcam, Cambridge, UK). Proteins were revealed and quantified with SuperSignal^® West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) on an ImageQuant imaging system (GE Healthcare Bio-Science AB, Uppsala, Sweden) or by fluorescence on an Odyssey imaging system (LI-COR, Lincoln, NE, USA). Uncropped Western blot bands can be found in Figures S4–S6.

Tétu P., Delyon J., André J., Reger de Moura C., Sabbah M., Ghanem G.E., Battistella M., Mourah S., Lebbé C, & Dumaz N. (2020). FGF2 Induces Resistance to Nilotinib through MAPK Pathway Activation in KIT Mutated Melanoma. Cancers, 12(5), 1062.

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Protein Expression Analysis by Western Blot

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Extraction of total protein was done using RIPA lysis buffer (NCM Biotech, China), followed by denaturation by adding 5× loading buffer. Proteins were separated by SDS-PAGE. Membranes was blocked by 5% BSA for 1 hour at room temperature. The membranes were placed in the primary antibodies: SNRPE (Thermo, PA5-96342, 1:1000), FGFR4 (Abcam, ab178396, 1:1000). Next, the membranes were placed in secondary antibodies after washing with TBST. Finally, membranes were observed in a Gel imaging System (TANON, Beijing, China). The images were quanti ed by Image J software. GAPDH or β-Actin was used as an internal reference.

Wu Q., Liao R., Miao C., Hasnat M., Li L., Sun L., Wang X., Yuan Z., Jiang Z., Zhang L, & Yu Q. (2024). Oncofetal SNRPE promotes HCC tumorigenesis by regulating the FGFR4 expression through alternative splicing. British journal of cancer, 131(1).

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Comprehensive Protein Expression Analysis

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Western blotting was performed as described previously.11 Primary antibodies against Wnt/β‐Catenin Activated Targets Antibody Sampler Kit, ERK1/2, p‐ERK1/2 (Thr202/Tyr204), AKT, p‐AKT (Ser473), p‐AKT (Thr308) (Cell Signaling Technology), SNAIL, FGFR4, Vimentin, Fibronectin and N/E‐cadherin (Abcam) were used under recommended dilution. GAPDH was used as an internal reference. The signals were detected using the SuperSignal West Dura Extended Duration Substrate Kit (Thermo Scientific Pierce, Rockford, IL, USA). The results were analyzed using the Image Lab software.

Yu T., Wang L., Li W., Zuo Q., Li M., Zou Q, & Xiao B. (2018). Downregulation of miR‐491‐5p promotes gastric cancer metastasis by regulating SNAIL and FGFR4. Cancer Science, 109(5), 1393-1403.

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Immunohistochemical Analysis of FGF23, Klotho, and TGFβ

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Twelve weeks after starting the induction of CRS, heart and kidney tissues were harvested from each group, fixed in 4% paraformaldehyde, and embedded in paraffin. Then 4 μm sections were prepared for immunostaining. After antigen retrieval by heating in citrate buffer (pH 6.0), the sections were incubated with primary antibodies overnight at 4° C. FGF23 (1:50, Santa Cruz, CA, USA), Klotho (1:50, R&D Systems, MN, USA), FGFR4 and TGFβ (1:50) (Abcam, MA, USA). The sections were incubated with an HRP-labeled secondary antibody. The Dako EVision+System-HRP (DAB) was used to visualize the indicated protein staining. Images were captured using an upright microscope (Olympus, Japan).

Hao H., Ma S., Zheng C., Wang Q., Lin H., Chen Z., Xie J., Chen L., Chen K., Wang Y., Huang X., Cao S., Liao W., Bin J, & Liao Y. (2021). Excessive fibroblast growth factor 23 promotes renal fibrosis in mice with type 2 cardiorenal syndrome. Aging (Albany NY), 13(2), 2982-3009.

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Western Blot Analysis of Key Signaling Proteins

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Samples containing equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinyl difluoride membranes. The membranes were blocked with 5% BSA at room temperature for 1 h, and then incubated overnight at 4° C with primary antibodies for the following targets: collagen I (1:1000, Bioss, Bejing, China), calcineurin A and FGFR4 (1:1000) (Abcam, MA, USA), vimentin, TGF-β, NFAT, p38 MAPK (1:1000) and phospho-p38 MAPK (1:500) (Cell Signaling Technology, MA, USA), p-GSK3α/β (1:500) and GSK3α/β (1:1000) (Santa Cruz, CA, USA), active-β-catenin (1:500) and β-catenin (1:1000) (Millipore, Darmstadt, Germany), Klotho and FGF23 (1:500) (R&D Systems, MN, USA), GAPDH (1:2000, Arigo, Shanghai, China). The reliability of the Klotho and FGF23 antibodies has been confirmed by previous study [6 (link), 70 (link)–72 (link)]. Then the membranes were incubated with Dylight 800-labeled secondary antibodies and reaction products were detected with a western blotting detection system (Infrared Imaging System, LI-COR, USA). Relative expression was quantified by densitometry with Image J Analysis Software (National Institutes of Health, Bethesda, MD).

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