Journal: Pharmaceutics

Article Title: Brain Delivery of IGF1R5, a Single-Domain Antibody Targeting Insulin-like Growth Factor-1 Receptor

doi: 10.3390/pharmaceutics14071452

Figure Lengend Snippet: SPR-derived kinetics and equilibrium dissociation constants for V H H-IGF1R interactions. 1 Determined at pH 7.4, 25 °C; 2 determined at pH 5.6, 37 °C.

Article Snippet: Ectodomains of human IGF1R (R&D Systems, Cat#391-GR-050), rhesus IGF1R (NRC Montreal, aa31-932), rat IGF1R (NRC Montreal, aa31-936) and mouse IGF1R (R&D Systems, Cat#6630-GR/CF-025) were amine coupled on CM5 sensor chips (Cytiva) at 10 μg/mL in 10 mM acetate pH 4.0 using an amine-coupling kit (Cytiva), resulting in approximately 1500–2000 response units (RUs) of each IGF1R ectodomain immobilized.

Techniques:

Journal: Pharmaceutics

Article Title: Brain Delivery of IGF1R5, a Single-Domain Antibody Targeting Insulin-like Growth Factor-1 Receptor

doi: 10.3390/pharmaceutics14071452

Figure Lengend Snippet: High-affinity binding of IGF1R5 V H Hs to IGF1R ectodomains. ( A ) SPR sensorgrams demonstrating wild-type IGF1R5 and humanized IGF1R5-H2 V H Hs binding to surface-immobilized human, rhesus, mouse and rat IGF1R (pH 7.4, 25 °C). V H H concentrations in flow ranged from 0.25 to 10 nM (IGF1R5) and from 1 to 25 nM (IGF1R5-H2). Kinetics and affinities were determined using multi-cycle kinetics (human, mouse, rat IGF1R) or single-cycle kinetics (rhesus IGF1R) analyses. ( B ) Sensorgrams demonstrating the binding of V H Hs to human and mouse IGF1R at acidic pH (pH 5.6, 37 °C). V H H concentrations in flow ranged from 0.25 to 10 nM (IGF1R5) and from 1 to 50 nM (IGF1R5-H2). Black lines: raw data; red lines: 1:1 binding model fitting.

Article Snippet: Ectodomains of human IGF1R (R&D Systems, Cat#391-GR-050), rhesus IGF1R (NRC Montreal, aa31-932), rat IGF1R (NRC Montreal, aa31-936) and mouse IGF1R (R&D Systems, Cat#6630-GR/CF-025) were amine coupled on CM5 sensor chips (Cytiva) at 10 μg/mL in 10 mM acetate pH 4.0 using an amine-coupling kit (Cytiva), resulting in approximately 1500–2000 response units (RUs) of each IGF1R ectodomain immobilized.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Cathepsin B regulates ovarian reserve quality and quantity via mitophagy by modulating IGF1R turnover

doi: 10.1101/2024.02.14.580410

Figure Lengend Snippet: a, Venn diagram of VC and i_Cat B -treated ovaries proteome. b , Principal Component Analysis (PCA) shows the difference between the VC and i_Cat B -treated ovaries proteome profiles. c, Relative abundance of differential proteins in VC and i_Cat B -treated ovaries presented as a heatmap. d, Distribution of all protein classes identified in VC and i_Cat B - treated ovaries according to biological process. e, A volcano plot showing P values versus fold changes of all proteins in VC and i_Cat B -treated ovaries. f , Schematic representation of insulin signalling pathway in oocytes. g, Western blot analysis of IGF1, IGF1R, pIGF1R, pAKT, pMTOR, Cat B, MVH and Tubulin from VC and i_Cat B -treated ovaries. h , Experimental regimen for in vitro reaction followed by mass spectrometry. i , Western blot analysis of in vitro digestion by Cat B. j , Schematic representation of invitro digested peptides of IGF1R identified in mass spectrometry. k , Experimental regimen for H1299 cell culture experiments. l, Western blot analysis of Cat B, IGF1R, and Actin from Control and Cat B-overexpressed H1299 cells. m, Western blot analysis of Cat B, IGF1R, and Actin from Control and i_Cat B -treated H1299 cells. VC, vehicle control; i_Cat B, an inhibitor of Cathepsin B.

Article Snippet: For in vitro reaction,1μg of purified IGF1R (R&D systems- 305-GR) was incubated with assay buffer 30μl [25 mM MES (pH 5)] in the presence or absence of purified Cat B [R&D systems- 953-CY, (500 ng)].

Techniques: Western Blot, In Vitro, Mass Spectrometry, Cell Culture, Control

Journal: bioRxiv

Article Title: Cathepsin B regulates ovarian reserve quality and quantity via mitophagy by modulating IGF1R turnover

doi: 10.1101/2024.02.14.580410

Figure Lengend Snippet: a, Experimental regime for intraperitoneal injection of Cathepsin B and IGF1R inhibitors. b, 21 dpp ovary sections from VC, i_Cat B, i_IGF1R and i_Cat B + i_IGF1R -treated females immunostained for TAp63α with hematoxylin counterstaining. c, Follicle counts at 21 dpp from VC, i_Cat B, i_IGF1R and i_Cat B + i_IGF1R -treated females. d, Types of follicles from 21 dpp from VC, i_Cat B, i_IGF1R and i_Cat B + i_IGF1R -treated females. VC, vehicle control; i_Cat B, an inhibitor of Cathepsin B; i_IGF1R, an inhibitor of IGF1R. The blue square indicates the follicle-enriched areas. **** P ≤ 0.0001, *** P ≤ 0.0002, ** P ≤ 0.02, * P ≤ 0.04, ns ≥ 0.1 unpaired t test. Error bars show mean ± SD. Non-significance P values are not represented in the figure. Scale bars for ovary sections are 100 μm.

Article Snippet: For in vitro reaction,1μg of purified IGF1R (R&D systems- 305-GR) was incubated with assay buffer 30μl [25 mM MES (pH 5)] in the presence or absence of purified Cat B [R&D systems- 953-CY, (500 ng)].

Techniques: Injection, Control

Journal: bioRxiv

Article Title: Cathepsin B regulates ovarian reserve quality and quantity via mitophagy by modulating IGF1R turnover

doi: 10.1101/2024.02.14.580410

Figure Lengend Snippet: a, Relative abundance of IGF1R pathway-related proteins in VC and i_Cat B-treated ovaries. b, Experimental regime for intraperitoneal injection of VC and i_Cat B -treated ovaries followed by JC1, mitotracker, lysotracker, and ROS experiments. c, Early follicles from VC and i_Cat B -treated ovaries were stained for JC1 and imaged at 488nm for JC-1 monomers and 565nm for dimers. d, Quantification of the signal intensity ratio of Dimer/Monomer. e, Early follicles from VC and i_Cat B -treated ovaries were stained for mitotracker green. f, Quantification of the signal intensity of mitotracker. g, Early follicles from VC and i_Cat B - treated ovaries were stained for lysotracker red. h, Quantification of the signal intensity of lysotracker. i, Early follicles from VC and i_Cat B -treated ovaries were stained for ROS. j, Quantification of the signal intensity of ROS. K, Experimental regime for intraperitoneal injection of VC and i_Cat B -treated ovaries followed by RT PCR experiments. l and m, Quantification of the relative fold change of BNIP3, TFAM gene expression in VC and i_Cat B -treated ovaries. VC, vehicle control; i_Cat B, an inhibitor of Cathepsin B. **** P ≤ 0.0001, ** P ≤ 0.006, * P ≤ 0.04, ns ≥ 0.01 unpaired t test. Error bars show mean ± SD. Non-significance P values are not represented in the figure. Scale bars for follicles are 10 μm.

Article Snippet: For in vitro reaction,1μg of purified IGF1R (R&D systems- 305-GR) was incubated with assay buffer 30μl [25 mM MES (pH 5)] in the presence or absence of purified Cat B [R&D systems- 953-CY, (500 ng)].

Techniques: Injection, Staining, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Control

Journal: bioRxiv

Article Title: Cathepsin B regulates ovarian reserve quality and quantity via mitophagy by modulating IGF1R turnover

doi: 10.1101/2024.02.14.580410

Figure Lengend Snippet: a, Representative images of early follicles from 10 dpp mice ovary immunostained for Cathepsin B (Red), IGF1R (green) and DNA (blue). b and c, Quantification of the signal intensity of Cathepsin B and IGF1R of early follicles at high and low intensity of Cathepsin B respectively. d, Representative images of late follicles from 10 dpp mice ovary immunostained for Cathepsin B (Red), IGF1R (green) and DNA (blue). e and f, Quantification of the signal intensity of Cathepsin B and IGF1R of late follicles at high and low intensity of Cathepsin B respectively. Cat B, Cathepsin B. **** P ≤ 0.0001, unpaired t test. Error bars show mean ± SD. Scale bars for follicles are 20 μm.

Article Snippet: For in vitro reaction,1μg of purified IGF1R (R&D systems- 305-GR) was incubated with assay buffer 30μl [25 mM MES (pH 5)] in the presence or absence of purified Cat B [R&D systems- 953-CY, (500 ng)].

Techniques: