huprot human proteome microarray  (Cambridge Protein Arrays)

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

Journal: Cell Death & Disease

Article Title: PAK6 rescues pathogenic LRRK2-mediated ciliogenesis and centrosomal cohesion defects in a mutation-specific manner

doi: 10.1038/s41419-024-07124-4

Figure Lengend Snippet: a Distribution of PAK6 candidate interactors according to their Z-score retrieved from a Human Proteome Microarray probed with recombinant full-length human PAK6. b A GO:BP analysis using gProfiler g:GOSt ( https://biit.cs.ut.ee/gprofiler/gost ) was performed for PAK6 candidate interactors with Z score >2.5 (left) and for PAK6 interactors annotated in PPI web-based tools PINOT, HIPPIE and MIST (PHM) (right). GO:BP terms with 2000 (array) and 1000 (PHM) term size were grouped into semantic categories. c Venn diagrams showing overlaps between the primary cilium proteome (GO:0005929, 640 genes) and the experimental (array) PAK6 interactome (left) or the literature-based (PHM) PAK6 interactome (right). d Protein network of overlapping PAK6 interactors with the primary cilium proteome (c) (including PAK6) obtained with STRING ( https://string-db.org/cgi/input?sessionId=b1S4T5BW27rz&input_page_show_search=on ); number of nodes: 11, number of edges: 11, average node degree: 2, average local clustering coefficient: 0.591, expected number of edges: 3, PPI enrichment P -value: 0.000502. Blue nodes are ciliary proteins present in the experimental PAK6 interactome (array) and grey nodes are those found in the literature-based PAK6 interactome. The interaction between LRRK2 and PAK6 identified in this study (blue) has been inserted manually.

Article Snippet: HuProt TM Human Proteome Microarray v4.0 was purchased from Cambridge Protein Arrays (Babraham Research Campus, Cambridge, UK) and employed to screen PAK6 interactor candidates following manufacturer’s instructions.

Techniques: Microarray, Recombinant