Journal: EBioMedicine

Article Title: Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

doi: 10.1016/j.ebiom.2015.05.028

Figure Lengend Snippet: Selective activation of FGFR1c/KLB complex by bFKB1 (A) A proposed model for FGFR1c–KLB–bispecific Ab (bFKB1) complex formation for signal activation. D1, D2 and D3 denote each of the Immunoglobulin-like domains in FGFR1 extra cellular domain (ECD). KD: kinase domain. (B) A predicted model for FGFR1c–KLB–FGF21 complex formation for signal activation. (C) GAL–ELK1 luciferase assay of human FGF21 and bFKB1 activity using FGFR1 –KO HEK293T cells generated by CRISPR/Cas9-mediated genome editing. Cells were transfected to express indicated receptors. h: human, c: cynomolgus monkey, and m: murine. Mean relative luciferase unit (RLU) ± SEM is shown. (D) GAL–ELK1 luciferase assay in L6 cells. Cells were co-transfected to express indicated receptors. Transfected cells were incubated with various concentrations of bFKB1, hFGF21, hFGF19 or R1MAb1. Mean fold change ± SEM in RLU is shown. (E) Western blot analysis of primary human adipocytes treated with indicated protein (hFGF21 (100 nM) or IgG (33 nM)) for 1 h. Each treatment was performed in duplicate. (F) Expression of UCP1 mRNA in primary human adipocytes treated with indicated protein at 30 nM for 48 h. N = 3. p < 0.005 (**). Data are presented as mean fold over vehicle control ± SEM.

Article Snippet: Antibodies used for western blot analysis were from Cell Signaling Technology: pFRS2a (T196) (#3864), pMEK1/2 (S217/221) (#9154), MEK (#9126), pERK1/2 (T202/204) (#4370), ERK1/2 (#4695), HSP90 (#4874), β-Actin (#5125), from abcam: UCP1 (ab10983), or from R&D Systems: KLB (AF2619).

Techniques: Activation Assay, Luciferase, Activity Assay, Generated, CRISPR, Transfection, Incubation, Western Blot, Expressing, Control

Journal: Hepatology Communications

Article Title: Profiling of cell‐free DNA methylation and histone signatures in pediatric NAFLD: A pilot study

doi: 10.1002/hep4.2082

Figure Lengend Snippet: Comparison of characteristics in children without (no NASH) versus children with NASH

Article Snippet: The patatin‐like phospholipase domain containing 3 (PNPLA3) rs738409, transmembrane 6 superfamily member 2 (TM6SF2) rs58542926, membrane bound O‐acyltransferase domain containing 7 (MBOAT7) rs641738, and klotho‐β (KLB) rs17618244 variants were genotyped by allelic discrimination using TaqMan 5′‐nuclease assays (Life Technologies).

Techniques: Comparison

Journal: Journal of pharmaceutical and biomedical analysis

Article Title: Noncompetitive immunoassay optimized for pharmacokinetic assessments of biologically active efruxifermin.

doi: 10.1016/j.jpba.2023.115402

Figure Lengend Snippet: Fig. 3. Flow chart of workflow for generation, purification, and conjugation of affinity purified chicken anti-EFXcore polyclonal antiserum.

Article Snippet: Following transfer to a nitrocellulose membrane, a polyclonal goat anti-human β-klotho antibody (R&D systems, cat # AF5889) was used, followed by an HRP-tagged rabbit anti-goat IgG secondary antibody (Invitrogen, cat. # 81–1620). β-actin was probed with a polyclonal rabbit anti-human β-actin primary antibody (Invitrogen, cat # PAI-183) followed by an HRP-tagged goat anti-rabbit IgG secondary antibody (Invitrogen, cat. # 31460).

Techniques: Purification, Conjugation Assay, Affinity Purification

Journal: Nature medicine

Article Title: Fibroblast growth factor 19 regulates skeletal muscle mass and ameliorates muscle wasting in mice.

doi: 10.1038/nm.4363

Figure Lengend Snippet: Figure 3 β-Klotho is essential for FGF19-induced muscle hypertrophy. Control and Klb mKO mice (8 weeks old) were treated with 0.1 mg/kg of FGF19 or vehicle for 7 d. (a) Muscle weight in grams (left) or in the percentage of body weight (right) of soleus, tibialis anterior (TA) and gastrocnemius (Gastro.) from mice treated with FGF19 or vehicle. (b) Frequency distribution of cross-sectional soleus-muscle fiber area and mean soleus fiber area. n.s., not significant (P = 0.1). (c) Percentage of slow-twitch (type I) and fast-twitch (type II) oxidative fibers in soleus muscles from control and Klb mKO mice. (d) Effect of FGF19 on type I and type II oxidative fiber area in soleus muscles from control and Klb mKO mice. n.s., not significant. n = 5 per group for control mice and n = 3 per group for Klb mKO mice. Data are mean ± s.e.m. Statistical analysis was done using a two- tailed Student’s t-test. *P < 0.05, **P < 0.01.

Article Snippet: The nitrocellulose membranes were incubated with β-Klotho-biotinylated primary antibody (R&D Systems, BAF-2619, 1:400) for 2 h at room temperature (RT).

Techniques: Control, Muscles, Two Tailed Test

Journal: EBioMedicine

Article Title: A novel GLP-1 and FGF21 dual agonist has therapeutic potential for diabetes and non-alcoholic steatohepatitis

doi: 10.1016/j.ebiom.2020.103202

Figure Lengend Snippet: Summary of the kinetic and affinity constants (KD) for the interaction between FGF21 mutants and β-Klotho using streptavidin (SA) biosensors.

Article Snippet: Rabbit Anti-Human β-Klotho monoclonal antibody (R&D Systems, Cat #MAB58891) or isotype control antibody (R&D Systems, Cat # AB-105-C), and anti-Rabbit IgG (PE) secondary antibody (R&D Systems, Cat # F0110) were used for β-Klotho expression.

Techniques:

Journal: EBioMedicine

Article Title: A novel GLP-1 and FGF21 dual agonist has therapeutic potential for diabetes and non-alcoholic steatohepatitis

doi: 10.1016/j.ebiom.2020.103202

Figure Lengend Snippet: Dual target GLP-1-Fc-FGF21 D1 functions through both GLP-1R and FGF21R/β-Klotho pathways. GLP-1-Fc-FGF21 D1, Fc-FGF21 S1 and Dulaglutide were investigated in cell based assays for their trans-activating activity. (a) ERK phosphorylation was measured in HEK293 cells expressing β-Klotho treated with native FGF21, GLP-1-Fc-FGF21 D1, Fc-FGF21 S1 or Dulaglutide. (b) cAMP secreted from HEK93 cells expressing GLP-1R treated with GLP-1-Fc-FGF21 D1, Fc-FGF21 S1 or dulaglutide. (c) HEK93 cells expressing GLP-1R and β-Klotho were treated with GLP-1-Fc-FGF21 D1, Fc-FGF21 S1, Dulaglutide or a 1:1 mixture of Fc-FGF21 S1 and Dulaglutide and the secreted cAMP were measured. (d) ERK phosphorylation was measured in HEK293 cells expressing GLP-1R and β-Klotho treated with GLP-1-Fc-FGF21 D1, Fc-FGF21 S1, Dulaglutide or a 1:1 mixture of Fc-FGF21 S1 and Dulaglutide. Data are showed as mean +/- standard errors of means, n =3.

Article Snippet: Rabbit Anti-Human β-Klotho monoclonal antibody (R&D Systems, Cat #MAB58891) or isotype control antibody (R&D Systems, Cat # AB-105-C), and anti-Rabbit IgG (PE) secondary antibody (R&D Systems, Cat # F0110) were used for β-Klotho expression.

Techniques: Activity Assay, Phospho-proteomics, Expressing

Journal: British Journal of Pharmacology

Article Title: Genetic fusion of human FGF21 to a synthetic polypeptide improves pharmacokinetics and pharmacodynamics in a mouse model of obesity

doi: 10.1111/bph.13499

Figure Lengend Snippet: The characterization of FGF21 and fusion proteins. The unmodified FGF21 and fusion proteins were assayed by (A) 12% SDS‐PAGE and (B) Western blotting with a rabbit anti‐human FGF21 antibody (lane 1: FGF21; lane 2: PsTag200‐FGF21; lane 3: PsTag400‐FGF21; lane 4: PsTag600‐FGF21). (C) CD spectra of unmodified FGF21 and fusion proteins. (D) IEF of FGF21 and PsTag600‐FGF21 (lane 1: PsTag600‐FGF21; lane 2: FGF21). (E) MALDI‐TOF mass spectrometry of PsTag600‐FGF21 (M+ and M2 + refer to the singly and doubly charged ionic species of PsTag600‐FGF21 respectively. (F) SEC‐HPLC in the presence of 150 mmol·L−1 sodium phosphate buffer (pH 7.0) resulted in a single peak with decreasing elution time for PsTag fusion proteins with increasing number of amino acid residues. (G) Binding affinities of FGF21 and PsTag fusion proteins to human β‐klotho were examined by direct binding elisa. (H) Cellular glucose uptake stimulated by native FGF21 and PsTag fused FGF21 in 3 T3‐L1 cells. n = 3. *P < 0.05 versus vehicle control. (I) 3T3‐L1 cells were treated with vehicle (lane 1), 10 nmol·L−1 FGF21 (lane 2), 10 nmol·L−1 PsTag200‐FGF21 (lane 3), 10 nmol·L−1 PsTag400‐FGF21 (lane 4) and 10 nmol·L−1 PsTag600‐FGF21 (lane 5) for 10 min. Phospho‐specific antibody was used to determine phosphorylation of ERK. (J) Pharmacokinetic plasma profile of native FGF21 and PsTag fusion proteins intravenously injected in C57BL/6 mice (n = 10 per group).

Article Snippet: Binding affinities of FGF21 and PsTag fusion proteins to human β‐klotho (58 890 KB, R&D) were examined by direct binding elisa .

Techniques: SDS Page, Western Blot, Mass Spectrometry, Binding Assay, Enzyme-linked Immunosorbent Assay, Injection