huprot human proteome microarray assays  (PEPperPRINT gmbh)

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

Journal: Nature Communications

Article Title: Capture of RNA-binding proteins across mouse tissues using HARD-AP

doi: 10.1038/s41467-024-52765-w

Figure Lengend Snippet: a Schematic of generating a pool of Cy5-labeled RNAs and hybridizing this pool with the human protein microarray. b Scatter plot showing the correlation of the fold change (Cy5 foreground signals over local background) of the HARD-AP identified RBPs ( n = 1447) in HEK293 cells. The fold change was calculated from the average of four independent protein spots on the two independent protein arrays (the same for c ). The Pearson correlation coefficient is given. c Distributions of the fold change and signal-noise-ratio (SNR) of indicated sets of proteins on the protein microarray (GO RBP n = 1365; HARD-AP RBP n = 1447; HARD-AP specific RBP n = 643). The buffer ( n = 320), BSA ( n = 80), GST ( n = 320), Ig A/G ( n = 720) at different concentrations were used as negative controls, and the Alexa 647 labeled IgG ( n = 80) was used as the positive control. d Pie chart showing the distribution of fold change (Cy5 signals over local background) of the HARD-AP identified RBPs ( n = 1447) and HARD-AP specific RBPs (n = 643) in HEK293 cells available on the protein microarray. e Top GO terms (molecular function and biological process) over-represent in HARD-AP RBPs with FC = 1-1.5 ( n = 745) or FC ≥ 1.5 ( n = 620) on two independent protein microarrays. The GO enrichment analysis used the two-sided Fisher’s exact test with the p -value adjusted using the Bonferroni correction for multiple testing. The number of proteins is labeled on each bubble. The color scale indicates the enrichment. f Images of Cy5-RNA incubation signal of selected proteins on the protein microarray. g Fold change of subunits of the Integrator, Exosome, Mediator, and 26S proteasome complex. Subunits with fold change over 1.5 were selected for plotting. Data are from four protein spots on two independent protein arrays and shown as the Mean ± SD. Source data for ( b – d , g ) are provided as a Source Data file.

Article Snippet: The HuProt Human proteome microarray contains over 21,000 GST-purified unique recombinant human proteins in yeast, including >81% of the canonically expressed proteins as defined by the Human Protein Atlas , .

Techniques: Labeling, Microarray, Positive Control, Incubation

Journal: Nature Communications

Article Title: Capture of RNA-binding proteins across mouse tissues using HARD-AP

doi: 10.1038/s41467-024-52765-w

Figure Lengend Snippet: a Volcano plot showing distributions of proteins captured by HARD beads compared to EGFP beads in indicated samples. The fold changes were calculated from means of the ion intensities of three independent biological samples. The significance (p) was determined using the two-tailed Student’s t-test and further adjusted using the Benjamini-Hochberg correction for multiple testing (p-adjust). b UpSet plot comparing RBPs identified in mESC and different mouse organs by HARD-AP. c Venn diagram comparing RBPs identified in different mouse samples by HARD-AP. d Matrix bubble plot showing enrichments of molecular function GO terms of in RBPs of indicated samples. The GO enrichment analysis used the two-sided Fisher’s exact test with the p-value adjusted using the Bonferroni correction for multiple testing. e Venn diagram comparing the mouse RBPome identified using HARD-AP (mouse RBPs_HARD) and all human RBPome (human RBPs_All) to their indicated orthologs. f Heatmap of the hierarchical clustering analysis using normalized ion intensities from indicated samples. g Top GO terms over-represent in tissue- and cell-enriched RBPs identified by HARD-AP. The GO enrichment analysis used the two-sided Fisher’s exact test with the p -value adjusted using the Bonferroni correction for multiple testing. h Relative levels of indicated proteins in different samples, which are calculated from the ion intensities of three independent biological samples. Data are means ± SD. HARD and EGFP represent proteins isolated by the HARD beads and EGFP beads respectively. i Western blot analysis showing the endogenous protein levels of Bcr, Prkar1a and Mylk3 in different mouse organs and mESC. This experiment was repeated once with similar results. j Western blot analysis for indicated proteins in indicated organ lysates after capture under indicated conditions. This experiment was repeated once with similar results. k Images of Cy5-RNA incubation signal of the human orthologs of Bcr, Prkar1a and Mylk3 on the protein microarray. Source data for ( a – c ) are provided as a Source Data file.

Article Snippet: The HuProt Human proteome microarray contains over 21,000 GST-purified unique recombinant human proteins in yeast, including >81% of the canonically expressed proteins as defined by the Human Protein Atlas , .

Techniques: Two Tailed Test, Isolation, Western Blot, Incubation, Microarray

Journal: Nature Communications

Article Title: Capture of RNA-binding proteins across mouse tissues using HARD-AP

doi: 10.1038/s41467-024-52765-w

Figure Lengend Snippet: a Left: Color matrix showing LIM domain protein on the protein microarray. Color scales represent the FC (Cy5 foreground signals over local background). Right: Image of Cy5-RNA incubation signal of the human CSRP1 on the protein microarray. b Relative levels of Csrp1 isolated from indicated mouse lysate using HARD beads and EGFP beads. Means ± SD are plotted; three independent biological samples were used for the analysis ( n = 3). The p -values were calculated using a two-tailed Student’s t -test. c Western blot analysis showing levels of Csrp1 isolated from the mouse brain and lung lysate using HARD beads and EGFP beads. This experiment was repeated once with similar results. d Heatmap of high-confidence CLIP-seq signals ± 2 kb around the center of peaks ( n = 10,028). Csrp1 IP: EBs expressing Csrp1-V5; Control: WT EBs. Two biologically independent replicates are shown. e Distribution of Csrp1 CLIP-seq signals ( n = 10,028) in different gene features. The average of the two biologically independent replicates is shown. f The top five GO terms associated with genes identified by CLIP-seq. The GO enrichment analysis used the two-sided Fisher’s exact test with the p -value adjusted using the Bonferroni correction for multiple testing. g Representative genome browser tracks showing normalized CLIP-seq and RNA-seq signals. h The top five enriched Csrp1-binding motifs on all target RNAs (target sequences = 6071, background sequences = 39,667). Motif enrichment significance was computed by HOMER (binomial test without adjustment). i Volcano plot showing distributions of genes differentially expressed in Csrp1 KO and WT embryoid bodies ( n = 22,625). The significance (p) was determined using the two-tailed Student’s t-test and further adjusted using the Benjamini-Hochberg correction for multiple testing (p-adjust). j Gene Set Enrichment Analysis of genes down-regulated in Csrp1 KO embryoid bodies compared to WT embryoid bodies. NES, normalized enrichment score. k The most enriched GO terms in significantly expressed genes in the Csrp1 KO embryoid bodies. The GO enrichment analysis used the two-sided Fisher’s exact test with the p -value adjusted using the Bonferroni correction for multiple testing. Source data for (b-c) are provided as a Source Data file.

Article Snippet: The HuProt Human proteome microarray contains over 21,000 GST-purified unique recombinant human proteins in yeast, including >81% of the canonically expressed proteins as defined by the Human Protein Atlas , .

Techniques: Microarray, Incubation, Isolation, Two Tailed Test, Western Blot, Expressing, Control, RNA Sequencing, Binding Assay

Journal: Bioactive Materials

Article Title: An intra articular injectable Mitocelle recovers dysfunctional mitochondria in cellular organelle disorders

doi: 10.1016/j.bioactmat.2024.09.021

Figure Lengend Snippet: Mitocelle can track and bind stressed mitochondria. (A) Schematic diagram of the human protein microarray analysis. (B) Mitocelle-interacting proteins were analyzed using the HuProt™ 3.1 human protein chip. The signal-to-noise ratio (SNR) for each spot was calculated as the ratio of the foreground-to-background signal. In addition, the GST signal intensity (red) was used for SNR normalization (left). A high-power image of NOX4 binding (white circles) is shown in the right panel. (C) Chord diagram visualizing the relationship between proteins with SNR > 1.0 and a list of mitochondrial, Golgi, and ER proteins. The SNR was calculated using the formula SNR = 20 log 10 (Is In −1 ), where ‘Is’ indicates the signal and ‘In’ indicates the noise. (D) Mouse chondrocytes treated with and without H 2 O 2 were analyzed by real-time live imaging of mitochondria (green) and Mitocelle (red) using a Celldiscoverer7 and an LSM900 confocal microscope. Intensity profiles of linear regions of interest are shown in the right panel. (E) To confirm mitochondrial dysfunction, we performed JC-1 staining and quantified JC-1 aggregates (red) and JC-1 monomers (green) as average intensities expressed in arbitrary units. Data are presented as means ± SD ( n = 5) and were assessed using one-way ANOVA with Bonferroni's test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Protein microarray data were generated using the HuProt Human Proteome Microarray v3.1 (CDI Laboratories, Mayaguez, Puerto Rico), which contains more than 21,000 unique and individually purified full-length human protein clones.

Techniques: Microarray, Binding Assay, Imaging, Microscopy, Staining

Journal: Bioactive Materials

Article Title: An intra articular injectable Mitocelle recovers dysfunctional mitochondria in cellular organelle disorders

doi: 10.1016/j.bioactmat.2024.09.021

Figure Lengend Snippet: Mitocelle interferes with the NOX4-p22phox interaction that contributes to ROS generation. (A) HuProt™ 3.1 Human Protein chip was used to analyze the effects of free Cy5 and Cy5-labeled Mitocelle (5 μg/mL) on Mitocelle-interacting proteins. The signal-to-noise ratio (SNR) of each point was calculated as the ratio of foreground to background signal. Additionally, a high-power image of p22phox binding (white circle) is shown in the right panel. (B) The SNR >1.0 proteins included 10 proteins known to be involved in ROS generation. (C) Schematic illustration of the principle of FRET and the acceptor photobleaching used for FRET measurements; CFP (donor), YFP (acceptor). (D) FRET detection by acceptor photobleaching. Chondrocytes were transfected for 24 h with vectors encoding YFP-NOX4 and CFP-p22phox, Mitocelles were applied, and transfection was continued for an additional 24 h. Fluorescence images were collected using YFP and CFP channels before and after photobleaching. To better show the changes in CFP fluorescence, pre- and post-bleaching CFP images are presented using pseudocolor. (E) FRET efficiency was measured after acceptor bleaching. Data are presented as means ± SD ( n = 10) and were assessed using (E) Mann-Whitney U test. ∗∗∗∗ P < 0.0001.

Article Snippet: Protein microarray data were generated using the HuProt Human Proteome Microarray v3.1 (CDI Laboratories, Mayaguez, Puerto Rico), which contains more than 21,000 unique and individually purified full-length human protein clones.

Techniques: Labeling, Binding Assay, Transfection, Fluorescence, MANN-WHITNEY

Journal: Chinese Medicine

Article Title: Polyphyllin I exerts anti-hepatocellular carcinoma activity by targeting ZBTB16 to activate the PPARγ/RXRα signaling pathway

doi: 10.1186/s13020-024-00984-0

Figure Lengend Snippet: ZBTB16 was identified as a direct target of PPI. A HuProt ™ 20 K Proteome microarray chip indicating the protein targets binding to PPI. The yellow arrow points to the location of the ZBTB16 protein. B Venn diagram of the protein targets binding to biotin and PPI. C , D GO and KEGG enrichment analysis of the specific binding proteins of PPI. E The signal intensity of the ZBTB16 protein in the HuProt ™ 20 K proteome microarray chip. F – H Changes in the expression of the ZBTB16 protein in HCC cells treated with PPI (n = 3; L, 2.5 μg/mL PPI; H, 5 μg/mL PPI). I SPR analysis of binding affinity between PPI and the ZBTB16 protein. J Molecular docking analysis of PPI binding to the ZBTB16 protein to predict the binding site. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Arrayit HuProt TM 20 K human proteome microarrays (CDI Laboratories, MD, USA) were used and the experiment conducted according to the operating standard for chip detection.

Techniques: Microarray, Binding Assay, Expressing