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

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: 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