Journal: eLife

Article Title: High-phytate/low-calcium diet is a risk factor for crystal nephropathies, renal phosphate wasting, and bone loss

doi: 10.7554/eLife.52709

Figure Lengend Snippet: Time-course analysis of serum levels of intact FGF23 for three consecutive phytate doses. Statistical significance was tested by repeated measure ANOVA between subjects and groups. All data are presented as the mean ± SD for each dose (n = 6 per dose).

Article Snippet: Rat intact PTH (Immutopics, San Clemente, CA, USA), human intact PTH (Abcam), soluble RANKL (Immundiagnostik, Bensheim, Germany), osteoprotegerin (Alpco Immunoassay, Salem, NH, USA), rat intact and C-terminal FGF23 (Elabscience, Houston, TX, USA), and human intact FGF23 (Elabscience) were measured using enzyme-linked immunosorbent assay.

Techniques:

Journal: eLife

Article Title: High-phytate/low-calcium diet is a risk factor for crystal nephropathies, renal phosphate wasting, and bone loss

doi: 10.7554/eLife.52709

Figure Lengend Snippet: ( A–C ) Time-course analysis of serum levels of intact PTH ( A ), 25(OH)D ( B ), and 1,25(OH) 2 D ( C ) in rats fed control, HP-LCa 2+ , and HP-HCa 2+ diets. For the early measurement of 25(OH)D ( B ) and 1,25(OH) 2 D, we pooled the sera from 2 to 3 rats. ( D–F ) Time-course analysis of renal CYP27B1 ( D ), CYP24A1 ( E ), and VDR ( F ) in rats fed control, HP-LCa 2+ , and HP-HCa 2+ diets. ( G ) Immunoblot analysis of renal αKlotho, NHERF1, NaPi-2a in rats fed control, HP-LCa 2+ , and HP-HCa 2+ diets. ( H ) The levels of renal proteins were quantified using ImageJ software (NIH, Bethesda, MD, USA). ( I ) Immunoblot analysis of calvarial FGF23 in rats fed control, HP-LCa 2+ , and HP-HCa 2+ diets. ( J ) The levels of total calvarial FGF23 were quantified using ImageJ software. All comparisons were conducted using two-way ANOVA with Tukey’s post hoc multiple comparison testing or the Kruskal–Wallis test ( A–C ) for non-parametric statistical analysis of data that did not have a normal distribution. Panels ( A–C ) failed the normality test, but no outliers were identified. All data are presented as the mean ± SD of each group (n = 4–6 per group). *, p<0.05; **, p<0.01; ***, p<0.001, compared with controls.

Article Snippet: Rat intact PTH (Immutopics, San Clemente, CA, USA), human intact PTH (Abcam), soluble RANKL (Immundiagnostik, Bensheim, Germany), osteoprotegerin (Alpco Immunoassay, Salem, NH, USA), rat intact and C-terminal FGF23 (Elabscience, Houston, TX, USA), and human intact FGF23 (Elabscience) were measured using enzyme-linked immunosorbent assay.

Techniques: Control, Western Blot, Software, Comparison

Journal: eLife

Article Title: High-phytate/low-calcium diet is a risk factor for crystal nephropathies, renal phosphate wasting, and bone loss

doi: 10.7554/eLife.52709

Figure Lengend Snippet: ( A ) The renal expression of downregulated genes associated with Ca 2+ and phosphate homeostasis in rats fed control or phytate-supplemented diets (n = 8 per group). ( B ) Results of immunoblot analyses and protein levels of renal aKlotho, NHERF1, and NaPi-2a after 2 weeks for rats fed control, HP- LCa 2+ , and HP-HCa 2+ diets. ( C ) The levels of renal proteins were quantified using ImageJ software (n = 6 per group). ( D, E ) Time course analysis of serum levels of intact FGF23 ( D ) and C-terminal FGF23 ( E ) in rats fed control, HP-LCa 2+ , and HP-HCa 2+ diets. ( F ) Representative immunohistochemical staining of EDTA-decalcified femur sections for FGF23 from rats fed control, HP-LCa 2+ , and HP-HCa 2+ diets. All data are presented as the mean ± SD of each group (n = 3–8 per group). *, p<0.05; **, p<0.01; ***, p<0.001, compared with controls.

Article Snippet: Rat intact PTH (Immutopics, San Clemente, CA, USA), human intact PTH (Abcam), soluble RANKL (Immundiagnostik, Bensheim, Germany), osteoprotegerin (Alpco Immunoassay, Salem, NH, USA), rat intact and C-terminal FGF23 (Elabscience, Houston, TX, USA), and human intact FGF23 (Elabscience) were measured using enzyme-linked immunosorbent assay.

Techniques: Expressing, Control, Western Blot, Software, Immunohistochemical staining, Staining

Journal: eLife

Article Title: High-phytate/low-calcium diet is a risk factor for crystal nephropathies, renal phosphate wasting, and bone loss

doi: 10.7554/eLife.52709

Figure Lengend Snippet:

Article Snippet: Rat intact PTH (Immutopics, San Clemente, CA, USA), human intact PTH (Abcam), soluble RANKL (Immundiagnostik, Bensheim, Germany), osteoprotegerin (Alpco Immunoassay, Salem, NH, USA), rat intact and C-terminal FGF23 (Elabscience, Houston, TX, USA), and human intact FGF23 (Elabscience) were measured using enzyme-linked immunosorbent assay.

Techniques: Control, Enzyme-linked Immunosorbent Assay, RIA Assay, Staining, Colorimetric Assay, Blocking Assay, Recombinant, In Vitro, Software

Journal: Cell Communication and Signaling : CCS

Article Title: The Roxadustat (FG-4592) ameliorates tubulointerstitial fibrosis by promoting intact FGF23 cleavage

doi: 10.1186/s12964-025-02175-2

Figure Lengend Snippet: FG-4592 lowers iFGF23 level in CKD rats and CKD patients. Parameters measured include ( A ) Serum intact fibroblast growth factor 23 (iFGF23) levels in CKD rats. B Serum total FGF23 levels in CKD rats. C Serum iFGF23 levels in CKD patients. D Serum total FGF23 levels in CKD patients. E Scatter plots with linear regression analysis demonstrating a significant correlation between blood urea nitrogen (BUN) and iFGF23 levels in CKD rats. F Scatter plots with linear regression analysis showing a significant correlation between Scr and iFGF23 levels in CKD rats. G Scatter plots with linear regression analysis revealing a significant correlation between iFGF23 levels and the percentage of fibrotic area in the kidney of CKD rats. H Scatter plots with linear regression analysis indicating a significant correlation between iFGF23 levels and Collagen 1 staining intensity in the kidney of CKD rats. I Scatter plots with linear regression analysis illustrating a significant correlation between iFGF23 levels and Fibronectin staining intensity in the kidney of CKD rats. J Scatter plots with linear regression analysis demonstrating a significant correlation between iFGF23 levels and α-SMA staining intensity in the kidney of CKD rats. Data are presented as means ± SD ( n = 5). Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The iFGF23 concentration was examined on plasma collected from rats under fed conditions using ELISA assays specific for rat iFGF23 (Quidel, cat#: 60–6800). iFGF23 and cFGF23 concentration were examined on plasma collected from rats under fed conditions using ELISA assays specific for rat total FGF23 (Quidel, cat#: 60–6300). iFGF23 and cFGF23 concentration were examined on plasma collected from CKD patients under fed conditions using ELISA assays specific for human iFGF23 or total FGF23 (Quidel, cat#: 60–6600 and cat#: 60–6100). iFGF23 ELISAs detect full-length, biologically active FGF23 that can exert phosphaturia, whereas total FGF23 ELISAs detect both iFGF23 and its C-terminal fragments and therefore provide a measure of the total amount of circulating plasma FGF23.

Techniques: Staining

Journal: Cell Communication and Signaling : CCS

Article Title: The Roxadustat (FG-4592) ameliorates tubulointerstitial fibrosis by promoting intact FGF23 cleavage

doi: 10.1186/s12964-025-02175-2

Figure Lengend Snippet: The renoprotective effects of FG-4592 were abrogated by FGF23 overexpression. Parameters measured include ( A ) Schematic diagram illustrating the experimental groups of rats: CKD + FG-4592 + vehicle and CKD + FG-4592 + rFGF23. B and C Renal function was assessed by measuring Scr and BUN levels. D mRNA expression levels of Col1a1 , Fn1 , and Acta2 in the renal cortex were quantified using real-time PCR. E Western blot analysis was performed to evaluate the protein expression of Collagen 1 and α-SMA, which reflect the extent of TIF. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as the loading control. F Representative images of PAS staining and the percentage of fibrotic area in kidney tissues. Scale bars: 50 μm. G Representative immunohistochemical images demonstrating the upregulation of Collagen 1 and α-SMA in the CKD + FG-4592 + rFGF23 group. Scale bars: 50 μm. Data are presented as means ± SD ( n = 5). Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The iFGF23 concentration was examined on plasma collected from rats under fed conditions using ELISA assays specific for rat iFGF23 (Quidel, cat#: 60–6800). iFGF23 and cFGF23 concentration were examined on plasma collected from rats under fed conditions using ELISA assays specific for rat total FGF23 (Quidel, cat#: 60–6300). iFGF23 and cFGF23 concentration were examined on plasma collected from CKD patients under fed conditions using ELISA assays specific for human iFGF23 or total FGF23 (Quidel, cat#: 60–6600 and cat#: 60–6100). iFGF23 ELISAs detect full-length, biologically active FGF23 that can exert phosphaturia, whereas total FGF23 ELISAs detect both iFGF23 and its C-terminal fragments and therefore provide a measure of the total amount of circulating plasma FGF23.

Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Staining, Immunohistochemical staining

Journal: Cell Communication and Signaling : CCS

Article Title: The Roxadustat (FG-4592) ameliorates tubulointerstitial fibrosis by promoting intact FGF23 cleavage

doi: 10.1186/s12964-025-02175-2

Figure Lengend Snippet: iFGF23 mediates FG-4592-induced inhibition of tubular cell fibrogenesis. HK-2 cells were pre-treated with TGF-β1 (10 ng/mL) for 24 h, followed by treatment with recombinant FGF23 (rFGF23, 50 ng/mL) or vehicle for an additional 24 h. Parameters measured include: A COL1A1 mRNA expression levels in HK-2 cells, as determined by quantitative PCR (qPCR). B TGFB1 mRNA expression levels in HK-2 cells, as determined by qPCR. C Collagen 1 protein expression levels in HK-2 cells, as assessed by Western blot analysis. D Schematic illustration of the co-culture system using UMR-106 cells and HK-2 cells in a Transwell setup. E mRNA expression levels of Fn1 , Acta2 , and Col1a1 in HK-2 cells, as quantified by qPCR. Data are presented as means ± SD ( n = 5). Statistical significance is denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: The iFGF23 concentration was examined on plasma collected from rats under fed conditions using ELISA assays specific for rat iFGF23 (Quidel, cat#: 60–6800). iFGF23 and cFGF23 concentration were examined on plasma collected from rats under fed conditions using ELISA assays specific for rat total FGF23 (Quidel, cat#: 60–6300). iFGF23 and cFGF23 concentration were examined on plasma collected from CKD patients under fed conditions using ELISA assays specific for human iFGF23 or total FGF23 (Quidel, cat#: 60–6600 and cat#: 60–6100). iFGF23 ELISAs detect full-length, biologically active FGF23 that can exert phosphaturia, whereas total FGF23 ELISAs detect both iFGF23 and its C-terminal fragments and therefore provide a measure of the total amount of circulating plasma FGF23.

Techniques: Inhibition, Recombinant, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Co-Culture Assay

Journal: Cell Communication and Signaling : CCS

Article Title: The Roxadustat (FG-4592) ameliorates tubulointerstitial fibrosis by promoting intact FGF23 cleavage

doi: 10.1186/s12964-025-02175-2

Figure Lengend Snippet: FG-4592 lowers iFGF23 levels through Furin-mediated cleavage. Parameters measured include ( A ) mRNA expression levels of Furin , Galnt3 , and Fam20c in bone tissues from the following groups: sham, CKD + vehicle, and CKD + FG-4592, as determined by quantitative PCR (qPCR). B Protein levels of HIF-1α, Furin, and intact fibroblast growth factor 23 (iFGF23) in bone tissues treated with FG-4592 or vehicle, as assessed by Western blot analysis. C Representative images of hematoxylin and eosin (HE) staining of bone tissues (scale bars: 20 μm), along with IHC staining for HIF-1α and iFGF23 (scale bars: 50 μm) and Furin (scale bars: 100 μm). D UMR-106 cells were pretreated with 10 nM 1,25(OH) 2 D 3 , and the expression of FGF23 mRNA was analyzed using real-time PCR. E Western blot analysis was performed to measure iFGF23 protein expression in UMR-106 cells. Cells were pretreated with indoxyl sulfate (IS, 0.5 mM) or vehicle, followed by treatment with FG-4592 (50 μM) or vehicle for 24 h. F Levels of iFGF23 and total FGF23 in the cell supernatant were quantified using an ELISA assay kit. G Western blot analysis was used to evaluate the protein expression of iFGF23 and Furin in UMR-106 cells. H Motif map of the Furin promoter region, highlighting the HIF-1 binding site. I ChIP assays demonstrated HIF-1 binding to the Furin promoter, which was enhanced by FG-4592 stimulation (50 μM for 24 h). J Protein expression levels of Furin and iFGF23 in UMR-106 cells following Furin siRNA interference, as determined by Western blot analysis. K Levels of iFGF23 and total FGF23 in the cell supernatant were measured using an ELISA assay kit after Furin siRNA interference. Data are presented as means ± SD ( n = 5). Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The iFGF23 concentration was examined on plasma collected from rats under fed conditions using ELISA assays specific for rat iFGF23 (Quidel, cat#: 60–6800). iFGF23 and cFGF23 concentration were examined on plasma collected from rats under fed conditions using ELISA assays specific for rat total FGF23 (Quidel, cat#: 60–6300). iFGF23 and cFGF23 concentration were examined on plasma collected from CKD patients under fed conditions using ELISA assays specific for human iFGF23 or total FGF23 (Quidel, cat#: 60–6600 and cat#: 60–6100). iFGF23 ELISAs detect full-length, biologically active FGF23 that can exert phosphaturia, whereas total FGF23 ELISAs detect both iFGF23 and its C-terminal fragments and therefore provide a measure of the total amount of circulating plasma FGF23.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: International Journal of Biological Sciences

Article Title: Molecular Interaction of Soluble Klotho with FGF23 in the Pathobiology of Aortic Valve Lesions Induced by Chronic Kidney Disease

doi: 10.7150/ijbs.92447

Figure Lengend Snippet: CKD increases FGF23 levels and decreases Klotho levels in the plasma and aortic valves of old mice. (A) Schematic diagram outlining the experimental protocols. Old mice (18-20 months) were on AIN-76A diet for 6 weeks and subjected to treatment with adenine and calcitriol from week 3 to week 5. (B) Representative images of von Kossa staining show valvular thickening and calcified nodule formation in the aortic valves of CKD mice. Scale bar = 100 μm. (C) CKD mice have higher FGF23 levels and lower Klotho levels in plasma in comparison to sham controls. (D) Representative images of immunofluorescence staining show increased FGF23 levels and reduced Klotho levels in aortic valves of CKD mice. Original magnification 40x. Scale bar = 100 µm. Quantitative data are expressed as mean ± SEM. n = 4 mice per group. * P < 0.05 versus sham controls.

Article Snippet: Endotoxin-free recombinant human FGF23 (2604), human Klotho (MAB5334), mouse Klotho (1819), neutralizing antibody against FGF23 (AF2604), and antibodies against YAP (867711) were purchased from R&D Systems, Minneapolis, MN.

Techniques: Clinical Proteomics, Staining, Comparison, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: Molecular Interaction of Soluble Klotho with FGF23 in the Pathobiology of Aortic Valve Lesions Induced by Chronic Kidney Disease

doi: 10.7150/ijbs.92447

Figure Lengend Snippet: FGF23 induces AVIC fibrosis and calcification. (A) Human AVICs from normal valves were treated with recombinant FGF23 in different concentrations for 72 hours. Representative immunoblots (left) and densitometric data (right) show that FGF23 upregulates the expression of inflammatory (ICAM-1 and VCAM-1), fibrogenic (collagen I and collagen IV) and osteogenic (RUNX2 and ALP) mediators in AVICs. (B) Representative images of Picrosirius Red (PSR) staining (upper) and Alizarin Red S staining (lower), along with corresponding spectrophotometric data, show that prolonged treatment with recombinant FGF23 (10 days or 14 days) induces collagen and calcium deposition in AVICs. Images were taken using a 10x objective. Scale bar = 100 µm. Quantitative data are expressed as mean ± SEM, n = 4 cell isolates from distinct donor valves in each group. * P < 0.05 versus untreated control.

Article Snippet: Endotoxin-free recombinant human FGF23 (2604), human Klotho (MAB5334), mouse Klotho (1819), neutralizing antibody against FGF23 (AF2604), and antibodies against YAP (867711) were purchased from R&D Systems, Minneapolis, MN.

Techniques: Recombinant, Western Blot, Expressing, Staining, Control

Journal: International Journal of Biological Sciences

Article Title: Molecular Interaction of Soluble Klotho with FGF23 in the Pathobiology of Aortic Valve Lesions Induced by Chronic Kidney Disease

doi: 10.7150/ijbs.92447

Figure Lengend Snippet: FGF23 is involved in mediating the pathobiological activities in AVICs of CAVD valves. (A) Immunoblots and densitometric data show that calcified aortic valves from patients with CAVD have higher levels of FGF23 and lower levels of Klotho in comparison to normal aortic valves. Values are means ± SEM. n = 4 aortic valve tissues from distinct donor in each group; * P < 0.05 versus normal aortic valves. N = normal. D = diseased. (B) Immunoblots and densitometric data show that human AVICs from diseased aortic valves have increased levels of FGF23 and decreased levels of Klotho. Values are means ± SEM. n = 4 cell isolates from distinct donor valves in each group. * P < 0.05 versus cells from normal valves. (C and D) Human AVICs from diseased valves were treated with lentivirus expressing FGF23 shRNA (100 nmol/L) or FGF23-neutralizing antibody (5 µg/mL) for 72 hours. Representative immunoblots and densitometric data show that AVICs treated with FGF23 shRNA or FGF23-neutralizing antibody have reduced levels of inflammatory, fibrogenic and osteogenic mediators. Values are mean ± SEM, n = 3 cell isolates from distinct donor valves in each group. * P < 0.05 versus control. Ctrl = control. N-AB = neutralizing antibody.

Article Snippet: Endotoxin-free recombinant human FGF23 (2604), human Klotho (MAB5334), mouse Klotho (1819), neutralizing antibody against FGF23 (AF2604), and antibodies against YAP (867711) were purchased from R&D Systems, Minneapolis, MN.

Techniques: Western Blot, Comparison, Expressing, shRNA, Control

Journal: International Journal of Biological Sciences

Article Title: Molecular Interaction of Soluble Klotho with FGF23 in the Pathobiology of Aortic Valve Lesions Induced by Chronic Kidney Disease

doi: 10.7150/ijbs.92447

Figure Lengend Snippet: FGF23 upregulates AVIC pathobiological activities via activation of the FGFR/YAP signaling pathway. (A) Human AVICs from normal valves were pre-treated with global FGFR inhibitor PRN1371 (1.0 µM) for 2 hours, followed by treatment with FGF23 (40 ng/mL) for 72 hours. Representative immunoblots and densitometric data show that FGF23 induces the production of inflammatory, fibrogenic and osteogenic mediators in AVICs via FGFR. (B) Total RNA-sequencing was applied to analyze gene expression in AVICs incubated in the presence or absence of recombinant FGF23. The heat map illustrates diferent levels of gene expression, and the KEGG data show upregulation of the Hippo signaling pathway in FGF23-treated cells (significant enrichment at P < 0.05, Fisher's exact test). (C) Cells were treated with recombinant FGF23 for different time. Representative immunoblots and densitometric data show that FGF23 induces YAP phosphorylation. (D) AVICs were treated with PRN1371 prior to FGF23 stimulation for 2 hours. Representative immunoblots and densitometric data show that global FGFR inhibitor suppresses FGF23-induced YAP phosphorylation. (F) Cells were treated with verteporfin (200 nM) for 2 hours prior to FGF23 stimulation. Representative immunoblots and densitometric data show that inhibition of YAP reduces inflammatory, fibrogenic, and osteogenic responses following FGF23 stimulation. Quantitative data are expressed as mean ± SEM. n = 3 or 4 cell isolates from distinct donor valves in each group. * P < 0.05 versus control and # P < 0.05 versus FGF23 alone.

Article Snippet: Endotoxin-free recombinant human FGF23 (2604), human Klotho (MAB5334), mouse Klotho (1819), neutralizing antibody against FGF23 (AF2604), and antibodies against YAP (867711) were purchased from R&D Systems, Minneapolis, MN.

Techniques: Activation Assay, Western Blot, RNA Sequencing, Gene Expression, Incubation, Recombinant, Phospho-proteomics, Inhibition, Control

Journal: International Journal of Biological Sciences

Article Title: Molecular Interaction of Soluble Klotho with FGF23 in the Pathobiology of Aortic Valve Lesions Induced by Chronic Kidney Disease

doi: 10.7150/ijbs.92447

Figure Lengend Snippet: Klotho interacts with FGF23 to suppress its effects in AVICs. (A) Human AVICs from normal valves were treated with varied concentrations of recombinant Klotho prior to the exposure to recombinant FGF23 for 72 hours. Representative immunoblots and densitometric data show that Klotho downregulates the effect of FGF23 in inducing inflammatory, fibrogenic and osteogenic mediators. (B) Representative images of Picrosirius Red (PSR) staining, Alizarin Red S staining and corresponding spectrophotometric data normalized by cell density show that recombinant Klotho suppresses collagen and calcium deposition in cells following prolonged exposure to FGF23. Original magnification 10x. Scale bar = 100 µm. (C) Representative immunoblots and densitometric data show that heat-denatured Klotho has no effect on FGF23-induced expression of inflammatory, fibrogenic and osteogenic mediators. (D) Recombinant FGF23 was incubated with recombinant Klotho for 24 hours. Representative immunoblot probed with anti-Klotho or anti-FGF23 identified corresponding free protein and the formation of complexes between these two proteins. (E) Pre-mixed Klotho-FGF23 has minimal effects on the expression of inflammatory, fibrogenic and osteogenic mediators in AVICs. Quantitative data are expressed as mean ± SEM. n = 3 or 4 cell isolates from distinct donor valves in each group. * P < 0.05 versus control and # P < 0.05 versus FGF23 alone.

Article Snippet: Endotoxin-free recombinant human FGF23 (2604), human Klotho (MAB5334), mouse Klotho (1819), neutralizing antibody against FGF23 (AF2604), and antibodies against YAP (867711) were purchased from R&D Systems, Minneapolis, MN.

Techniques: Recombinant, Western Blot, Staining, Expressing, Incubation, Control

Journal: Journal of Biological Chemistry

Article Title: Regulation of Fibroblast Growth Factor-23 Signaling by Klotho

doi: 10.1074/jbc.c500457200

Figure Lengend Snippet: FIGURE 2. FGF23 preferentially binds to the Klotho-FGFR complex. Agarose beads bound to FGFR, Klotho, or the Klotho-FGFR complex were incubated with conditioned medium containing mouse FGF23(R179Q) and probed with FGF23, FGFR (anti-V5 anti- body), or Klotho by immunoblotting.

Article Snippet: Thewashed beadswere suspended in SDS-sample loading buffer and subjected to immunoblot analysis using anti-V5 antibody, KM2119, or anti-FGF23 antibody (R&D Systems).

Techniques: Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Regulation of Fibroblast Growth Factor-23 Signaling by Klotho

doi: 10.1074/jbc.c500457200

Figure Lengend Snippet: FIGURE 3. FGF23 requires Klotho to activate FGF signaling. A, 293 cells or Klotho- expessing 293 cells (293KL) were stimulated with various concentration of recombinant human FGF23 or basic FGF (100 ng/ml) for 15 min. Activation of FGF signaling was determined by immunoblot analysis using anti-phospho-FRS2a antibody (p-FRS2a), anti-phospho-ERK1/2 antibody (p-ERK1/2), or anti-ERK1/2 antibody (ERK1/2). B, 293KL cells or 293KLDTM cells were stimulated with the conditioned medium containing mouseFGF23(R179Q)(10ng/ml)for15min.Thesamevolumeofconditionedmedium frommock-transfected293cellswasusedasanegativecontrol(FGF23).C,detectionof endogenousFGFRsboundtoKlothoin293KLcells.Lysatesof293,293KL,and293KLDTM cells were immunoprecipitated with Klotho using anti-FLAG antibody and then immu- noblotted with antibodies against FGFR1, FGFR2, FGFR3, and Klotho. Lysates of 293 cells transfected with expression vectors for mouse FGFR1c, FGFR2c, and FGFR3c were immu- noprecipitated with anti-V5 antibody and used as positive controls for immunoblotting of FGFR1, FGFR2, and FGFR3, respectively. D, a model for interaction between Klotho, FGFR, FGF23, and FGF signaling.

Article Snippet: Thewashed beadswere suspended in SDS-sample loading buffer and subjected to immunoblot analysis using anti-V5 antibody, KM2119, or anti-FGF23 antibody (R&D Systems).

Techniques: Concentration Assay, Recombinant, Activation Assay, Western Blot, Immunoprecipitation, Transfection, Expressing

Journal: Journal of Biological Chemistry

Article Title: Regulation of Fibroblast Growth Factor-23 Signaling by Klotho

doi: 10.1074/jbc.c500457200

Figure Lengend Snippet: FIGURE 4. Klotho functions as a regulator of FGF23 signaling in various types of cells. PC12 and HeLa cells were infected with adenovirous expressing the full-length Klotho or GFP as a negative control and then stimulated with mouse FGF23 (R179Q) for 15 min. CHO cells and Klotho-expressing CHO cells (CHOKL) were also stimulated in the same way. Cell lysates were immunoblotted with anti-Klotho antibody (KM2119) to con- firm Klotho expression. Activity of FGF signaling was analyzed in the same way as Fig. 3.

Article Snippet: Thewashed beadswere suspended in SDS-sample loading buffer and subjected to immunoblot analysis using anti-V5 antibody, KM2119, or anti-FGF23 antibody (R&D Systems).

Techniques: Infection, Expressing, Negative Control, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: A New Approach to the Quantification of Fibroblast Growth Factor 23—An Array Surface Plasmon Resonance Imaging Biosensor

doi: 10.3390/ijms242015327

Figure Lengend Snippet: Dependence of the SPRi signal on FGF23 antibody concentration. The used concentration of the FGF23-αKlotho complex was 40:169.2 pg/mL; pH = 7.4. “Standard deviation (SD)” was calculated from 12 individual measurements.

Article Snippet: The monoclonal mouse anti-human FGF23 antibody (R&D Systems) was a receptor that bound the FGF23 protein from samples to the chip’s surface.

Techniques: Concentration Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: A New Approach to the Quantification of Fibroblast Growth Factor 23—An Array Surface Plasmon Resonance Imaging Biosensor

doi: 10.3390/ijms242015327

Figure Lengend Snippet: Dependence of the SPRi signal on the FGF23 concentration. The used FGF23 antibody concentration is 25 ng/mL; pH = 7.4. SD was calculated from 12 individual measurements.

Article Snippet: The monoclonal mouse anti-human FGF23 antibody (R&D Systems) was a receptor that bound the FGF23 protein from samples to the chip’s surface.

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: A New Approach to the Quantification of Fibroblast Growth Factor 23—An Array Surface Plasmon Resonance Imaging Biosensor

doi: 10.3390/ijms242015327

Figure Lengend Snippet: Dependence of the SPRi signal on the FGF23 concentration with the addition of αKlotho protein. The used FGF23 antibody concentration is 25 ng/mL; pH = 7.4. SD was calculated from 12 individual measurements.

Article Snippet: The monoclonal mouse anti-human FGF23 antibody (R&D Systems) was a receptor that bound the FGF23 protein from samples to the chip’s surface.

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: A New Approach to the Quantification of Fibroblast Growth Factor 23—An Array Surface Plasmon Resonance Imaging Biosensor

doi: 10.3390/ijms242015327

Figure Lengend Snippet: Influence of the αKlotho protein on the detected FGF23 concentration. SD was calculated from 12 individual measurements.

Article Snippet: The monoclonal mouse anti-human FGF23 antibody (R&D Systems) was a receptor that bound the FGF23 protein from samples to the chip’s surface.

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: A New Approach to the Quantification of Fibroblast Growth Factor 23—An Array Surface Plasmon Resonance Imaging Biosensor

doi: 10.3390/ijms242015327

Figure Lengend Snippet: Influence of the time on the formation of the FGF23-αKlotho complex. SD was calculated from 12 individual measurements.

Article Snippet: The monoclonal mouse anti-human FGF23 antibody (R&D Systems) was a receptor that bound the FGF23 protein from samples to the chip’s surface.

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: A New Approach to the Quantification of Fibroblast Growth Factor 23—An Array Surface Plasmon Resonance Imaging Biosensor

doi: 10.3390/ijms242015327

Figure Lengend Snippet: The influence of the potential interferents on the determination of the FGF23 with array SPRi biosensor. SD was calculated from 12 individual measurements.

Article Snippet: The monoclonal mouse anti-human FGF23 antibody (R&D Systems) was a receptor that bound the FGF23 protein from samples to the chip’s surface.

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: A New Approach to the Quantification of Fibroblast Growth Factor 23—An Array Surface Plasmon Resonance Imaging Biosensor

doi: 10.3390/ijms242015327

Figure Lengend Snippet: Precision and accuracy of the FGF23 SPRi biosensor. SD and RSD were calculated from 36 individual measurements.

Article Snippet: The monoclonal mouse anti-human FGF23 antibody (R&D Systems) was a receptor that bound the FGF23 protein from samples to the chip’s surface.

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: A New Approach to the Quantification of Fibroblast Growth Factor 23—An Array Surface Plasmon Resonance Imaging Biosensor

doi: 10.3390/ijms242015327

Figure Lengend Snippet: Precision and recovery of the FGF23 SPRi biosensor tested in a plasma sample after the addition of a 25 pg/mL spike. SD and RSD were calculated from 12 individual measurements.

Article Snippet: The monoclonal mouse anti-human FGF23 antibody (R&D Systems) was a receptor that bound the FGF23 protein from samples to the chip’s surface.

Techniques: Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: A New Approach to the Quantification of Fibroblast Growth Factor 23—An Array Surface Plasmon Resonance Imaging Biosensor

doi: 10.3390/ijms242015327

Figure Lengend Snippet: Formation of the FGF23 SPRi biosensor layers, i.e., bare gold, cysteamine, FGF23 antibody, and FGF23-αKlotho complex, captured via SEM. Used parameters: 12.5 or 15 kV, and 100,000× magnification.

Article Snippet: The monoclonal mouse anti-human FGF23 antibody (R&D Systems) was a receptor that bound the FGF23 protein from samples to the chip’s surface.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A New Approach to the Quantification of Fibroblast Growth Factor 23—An Array Surface Plasmon Resonance Imaging Biosensor

doi: 10.3390/ijms242015327

Figure Lengend Snippet: The comparison of the results of FGF23 concentration measurements performed with the SPRi biosensor and ELISA in blood plasma samples. SD was calculated based on 12 measurements for the SPRi biosensor and 2 for the ELISA.

Article Snippet: The monoclonal mouse anti-human FGF23 antibody (R&D Systems) was a receptor that bound the FGF23 protein from samples to the chip’s surface.

Techniques: Comparison, Concentration Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: A New Approach to the Quantification of Fibroblast Growth Factor 23—An Array Surface Plasmon Resonance Imaging Biosensor

doi: 10.3390/ijms242015327

Figure Lengend Snippet: Scheme of the measuring system of the SPRi apparatus and an overview diagram of the FGF23 biosensor.

Article Snippet: The monoclonal mouse anti-human FGF23 antibody (R&D Systems) was a receptor that bound the FGF23 protein from samples to the chip’s surface.

Techniques: