Journal: Communications Medicine

Article Title: Preclinical characterization of an active immunotherapy targeting calcitonin gene-related peptide

doi: 10.1038/s43856-025-00870-2

Figure Lengend Snippet: Proteome and peptide screens were used to evaluate potential off-target binding of affinity-purified p4796kb antibodies. a Manhattan plot displays results from the HuProt Human Proteome Microarray screen of antibody binding. Colors represent each block from the array, containing approximately 1000 proteins each. Spots represent individual protein intensity score from array. Hits are annotated with protein names. b Manhattan plot from peptide counter screen of 21 putative hits from proteome screen. Overlapping 15mer peptides were synthesized and printed on arrays. Spots represent individual peptide intensity scores from array. Peptide sequences of hits are displayed. Sera from guinea pigs immunized with p4796kb were tested for binding to putative hits from screens ( c ) and calcitonin family ( d ) measured by ELISA. Pre-immune sera were used as controls. Data are presented as means +/− SEM; n = 3. *** p < 0.0001.

Article Snippet: Binding of p4796kb-derived sera to 3 hits from the HuProt™ microarray screen (HSP90 beta protein, StressMarq Bioscience, SPR-102B; AKR1B10 protein, MyBioSource, MBS203315; PTPRD protein, Acro, PTD-H52H9) and to other propeptides that belong to the calcitonin/CGRP peptide family, including recombinant adrenomedullin (ADM, MyBioSource, MBS2012013), recombinant adrenomedullin 2 (ADM2, MyBioSource, MBS2123890), synthetic amylin (Abcam, ab142398), and recombinant calcitonin (Abcam, ab153793), were further evaluated.

Techniques: Binding Assay, Affinity Purification, Microarray, Blocking Assay, Synthesized, Enzyme-linked Immunosorbent Assay

Journal: Cell Research

Article Title: Tau is a receptor with low affinity for glucocorticoids and is required for glucocorticoid-induced bone loss

doi: 10.1038/s41422-024-01016-0

Figure Lengend Snippet: a The schematic diagram of human proteome microarray, which contains over 20,000 individual proteins printed in duplicate, to identify binding partners of high-dose and low-dose dexamethasone, respectively. b , c Human proteome microarray analysis reveals the proteins (in blue) which bind to both low and high-dose dexamethasone, and proteins (in green) which selectively bind to high-dose dexamethasone. d Principle of DARTS assay for the isolation of proteins protected from degradation by dexamethasone. e Dexamethasone protects two groups of protein bands (highlighted in black rectangle) from degradation in DARTS using whole cell lysate from dexamethasone-treated Raw264.7 cells coupled with Coomassie blue staining. f Molecular weight (MW) plot of putative high-dose dexamethasone-binding proteins identified by human proteome microarray analysis. g The protective effects of serial doses of dexamethasone on Tau and GR from digestion by protease are evaluated by DARTS coupled with immunoblotting. GAPDH is resistant to protease under the condition and serves as a loading indicator. Representative image is shown ( n = 3). h Quantification of Tau and GR stability treated with serial dosages of dexamethasone assayed by DARTS ( n = 3). i The interaction between dexamethasone and Tau, assayed by solid phase binding. 10 mM Tau was coated to the plate, and a serial dilution of biotin-labeled dexamethasone was added, followed by incubation with HRP-labeled Streptavidin and its substrate ( n = 3). Inset shows the Scatchard plot analysis for K D value calculation. j – p One-step kinetic SPR assay for binding of Tau to different GCs, as indicated. q qRT-PCR analysis of Tau mRNA levels in different tissues, as indicated ( n = 3). r Double-immunoflurorescence staining of femur section using antibodies against Tau (green) and TRAP, osteocalcin (OCN) and sclerostin (SOST) (red). DAPI stains nuclei. Arrows indicate positive staining cells. Scale bar = 20 µm. BM, bone marrow. Data are means ± SD in h , i , q .

Article Snippet: HuProt human proteome microarray version 4.0 (HuProt TM , CDI Laboratories), which is composed of ~ 20,000 human FL proteins with N-terminal glutathione S-transferase tag was used to isolate dexamethasone-binding proteins.

Techniques: Microarray, Binding Assay, Isolation, Staining, Molecular Weight, Western Blot, Serial Dilution, Labeling, Incubation, SPR Assay, Quantitative RT-PCR