fgfr1 phosphorylation levels  (Proteintech)

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Posttranslational Modification Defects in Fibroblast Growth Factor Receptor 1 as a Reason for Normosmic Isolated Hypogonadotropic Hypogonadism

doi: 10.1155/2020/2358719

Figure Lengend Snippet: The correlation of genotypes and phenotypes between proband nIHH and reported Kallmann patient.

Article Snippet: Subsequently, the cells were subjected to immunoblotting with anti-p-FGFR1 (1 : 5000, Abcam), anti-FGFR1 (1 : 3500, Abcam), anti-p-ERK (1 : 1000, Cell Signaling Technology), anti-ERK1/2 (1 : 1000, Cell Signaling Technology), anti-p-Akt (1 : 1000, Cell Signaling Technology), anti-Akt (1 : 1000, Cell Signaling Technology), anti-p-STAT3 (1 : 1000, Cell Signaling Technology), anti-STAT3 (1 : 1000, Cell Signaling Technology), and anti-GAPDH (1 : 7000, Proteintech) to observe the expression of the FGFR1 phosphorylation levels as well as the relative phosphorylation levels of the downstream signaling molecules.

Techniques: Mutagenesis

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Posttranslational Modification Defects in Fibroblast Growth Factor Receptor 1 as a Reason for Normosmic Isolated Hypogonadotropic Hypogonadism

doi: 10.1155/2020/2358719

Figure Lengend Snippet: Pedigrees of the proband with FGFR1 and CEP290 mutation. (a) The mutation results in both FGFR1 (A) and CEP290 (B) from all family members. Arrows: mutation sites. (b) Pedigrees of the proband. Circles: females; squares: males; arrows: proband. (c) The domain structures of both FGFR1 (A) and CEP290 (B) with two mutant sites, respectively. SP: signal peptide; HB: binding domain for heparin or heparin sulfate proteoglycan; PLC sites: interaction with PLC gamma; NB: nuclear-binding domain; TK1/2: tyrosine kinase subdomain 1/2; Ig I, Ig II, and Ig III: three Ig-like domains; TM: transmembrane domain.

Article Snippet: Subsequently, the cells were subjected to immunoblotting with anti-p-FGFR1 (1 : 5000, Abcam), anti-FGFR1 (1 : 3500, Abcam), anti-p-ERK (1 : 1000, Cell Signaling Technology), anti-ERK1/2 (1 : 1000, Cell Signaling Technology), anti-p-Akt (1 : 1000, Cell Signaling Technology), anti-Akt (1 : 1000, Cell Signaling Technology), anti-p-STAT3 (1 : 1000, Cell Signaling Technology), anti-STAT3 (1 : 1000, Cell Signaling Technology), and anti-GAPDH (1 : 7000, Proteintech) to observe the expression of the FGFR1 phosphorylation levels as well as the relative phosphorylation levels of the downstream signaling molecules.

Techniques: Mutagenesis, Binding Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Posttranslational Modification Defects in Fibroblast Growth Factor Receptor 1 as a Reason for Normosmic Isolated Hypogonadotropic Hypogonadism

doi: 10.1155/2020/2358719

Figure Lengend Snippet: The bioinformatic assays of the mutations of FGFR1 and CEP290 . (a, b) (A) Conservation analysis of the two mutant sites via multiple sequence alignment. Amino acid in red color are the substitutive amino acid. Asterisks represent a high score of conservation degree. (B) The conservation degree of FGFR1 and CEP290 calculated by WebLogo software. The overall stack height represents the sequence conservation at that position, while the symbol height within the stack indicates the relative frequency of each amino or nucleic acid at that position. (c) The crystal structure of the mutation E670K upon the catalytic (tyrosine kinase) cytosolic domain of FGFR1 and the maps of the coiled-coil domain within mutant and WT CEP290. α -Helices, β -strands, and loops are colored cyan, red, and pink, respectively. Nitrogen and oxygen are colored blue and red, respectively. The mutation sites are labeled with yellow sticks. The phosphorylation site of Tyr-653 and Tyr-638 are shown as green sticks. The activation site is shown as grey sticks. Arrows point to the magnified pictures of selected residues. These structural images are shown using PyMOL.

Article Snippet: Subsequently, the cells were subjected to immunoblotting with anti-p-FGFR1 (1 : 5000, Abcam), anti-FGFR1 (1 : 3500, Abcam), anti-p-ERK (1 : 1000, Cell Signaling Technology), anti-ERK1/2 (1 : 1000, Cell Signaling Technology), anti-p-Akt (1 : 1000, Cell Signaling Technology), anti-Akt (1 : 1000, Cell Signaling Technology), anti-p-STAT3 (1 : 1000, Cell Signaling Technology), anti-STAT3 (1 : 1000, Cell Signaling Technology), and anti-GAPDH (1 : 7000, Proteintech) to observe the expression of the FGFR1 phosphorylation levels as well as the relative phosphorylation levels of the downstream signaling molecules.

Techniques: Mutagenesis, Sequencing, Software, Labeling, Activation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Posttranslational Modification Defects in Fibroblast Growth Factor Receptor 1 as a Reason for Normosmic Isolated Hypogonadotropic Hypogonadism

doi: 10.1155/2020/2358719

Figure Lengend Snippet: Analysis of subcellular localization and deglycosylation in FGFR1 groups. (a) Subcellular localization of FGFR1 in HEK293 cells. HEK293 cells were transfected with empty vector (EV), FGFR1 (WT), or mutant FGFR1 (p. E670K), and protein localization was observed by fluorescence microscopy. FGFR1 was detected using an anti-FGFR1 antibody followed by secondary antibodies conjugated with Alexa Fluor 555 (red). Nuclei were visualized by DAPI. Original magnification: 600x. (b) Deglycosylation in FGFR1 groups with Endo H and PNGase F. Overall expression levels of FGFR1 in distinct groups were judged from the PNGase F treatments and were normalized to their GAPDH levels, respectively. Maturation analysis was determined from the Endo H-treated groups; the upper band represents the fully glycosylated mature form while the lower band stands for an immature or a core glycosylated form. Percentage of the mature band by density calculations was used to measure the maturation degrees with the groups. Both the results represent the ratio between mutant and WT. EV: empty vector; UT: untreated; E: Endo H-treated; P: PNGase F treated. Arrows point to the molecular weight.

Article Snippet: Subsequently, the cells were subjected to immunoblotting with anti-p-FGFR1 (1 : 5000, Abcam), anti-FGFR1 (1 : 3500, Abcam), anti-p-ERK (1 : 1000, Cell Signaling Technology), anti-ERK1/2 (1 : 1000, Cell Signaling Technology), anti-p-Akt (1 : 1000, Cell Signaling Technology), anti-Akt (1 : 1000, Cell Signaling Technology), anti-p-STAT3 (1 : 1000, Cell Signaling Technology), anti-STAT3 (1 : 1000, Cell Signaling Technology), and anti-GAPDH (1 : 7000, Proteintech) to observe the expression of the FGFR1 phosphorylation levels as well as the relative phosphorylation levels of the downstream signaling molecules.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Fluorescence, Microscopy, Expressing, Molecular Weight

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Posttranslational Modification Defects in Fibroblast Growth Factor Receptor 1 as a Reason for Normosmic Isolated Hypogonadotropic Hypogonadism

doi: 10.1155/2020/2358719

Figure Lengend Snippet: Functional analysis of FGFR1 in the mutant and WT groups in vitro. (a) Gene expression analysis of FGFR1 in the mutant and WT groups by qPCR. HEK293 cells were transiently transfected with WT, mutant FGFR1 plasmid, or empty vector for RNA extraction, using RT-PCR and real-time quantitative PCR to detect the FGFR1 mRNA expression. (b–d) Gene and cell surface expression analysis of FGFR1 and its downstream signaling. (b) Using RT-PCR and qPCR for the FOS gene expression analysis in the FGF8-induced mutant and WT groups. (c) The phosphorylation levels of WT and mutant FGFR1 and the affected signal pathways tested by western blotting in groups (A). Quantitative analyses of FGFR1 phosphorylation levels (Y653) (B), the total FGFR1 relative level (C), FGF8-induced ERK1/2 (D), and Akt (E) phosphorylation levels are shown with a bar chart. (d) Analysis on the FGFR1-affected JAK/STAT3 pathways by western blotting in groups (A). Quantitative analysis of the STAT3 phosphorylation levels (B). Shown is the meanpercentage ± SD of three biological replicates ( p < 0.01 by Student's t -test).

Article Snippet: Subsequently, the cells were subjected to immunoblotting with anti-p-FGFR1 (1 : 5000, Abcam), anti-FGFR1 (1 : 3500, Abcam), anti-p-ERK (1 : 1000, Cell Signaling Technology), anti-ERK1/2 (1 : 1000, Cell Signaling Technology), anti-p-Akt (1 : 1000, Cell Signaling Technology), anti-Akt (1 : 1000, Cell Signaling Technology), anti-p-STAT3 (1 : 1000, Cell Signaling Technology), anti-STAT3 (1 : 1000, Cell Signaling Technology), and anti-GAPDH (1 : 7000, Proteintech) to observe the expression of the FGFR1 phosphorylation levels as well as the relative phosphorylation levels of the downstream signaling molecules.

Techniques: Functional Assay, Mutagenesis, In Vitro, Expressing, Transfection, Plasmid Preparation, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Posttranslational Modification Defects in Fibroblast Growth Factor Receptor 1 as a Reason for Normosmic Isolated Hypogonadotropic Hypogonadism

doi: 10.1155/2020/2358719

Figure Lengend Snippet: A relevance between GnRH-deficiency-related signaling network and several known disease-causing genes of olfactory dysfunction by using STRING software. The protein-protein interaction network encoded by pathogenic genes known to cause KS including FGFR1 (colored blue) were correlated with CEP290-related protein-protein network involving olfactory dysfunction via several pathways containing the factors of FGF8, NTRK2, AHI1, and IFT88.

Article Snippet: Subsequently, the cells were subjected to immunoblotting with anti-p-FGFR1 (1 : 5000, Abcam), anti-FGFR1 (1 : 3500, Abcam), anti-p-ERK (1 : 1000, Cell Signaling Technology), anti-ERK1/2 (1 : 1000, Cell Signaling Technology), anti-p-Akt (1 : 1000, Cell Signaling Technology), anti-Akt (1 : 1000, Cell Signaling Technology), anti-p-STAT3 (1 : 1000, Cell Signaling Technology), anti-STAT3 (1 : 1000, Cell Signaling Technology), and anti-GAPDH (1 : 7000, Proteintech) to observe the expression of the FGFR1 phosphorylation levels as well as the relative phosphorylation levels of the downstream signaling molecules.

Techniques: Software