Polyubiquitination analysis upon GSK3 inhibition in HeLa cell extracts
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Emil Karaulanov
PROVIDER: E-GEOD-50629 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:HeLa cell extracts with or without GSK3 enzyme inhibition were assayed using protein microarrays in order to detect GSK3-dependent changes in protein polyubiquitination.
2014-05-15 | GSE50629 | GEO
Project description:Type 1 diabetes mellitus (T1DM) results from immune mediated destruction of pancreatic beta cells. However, clinical and immunologic phenotypes of T1DM are variable. Several auto-antibodies including GADA, IA-2A, and ZnT8A, were identified in T1DM, but the prevalence of these auto-antibodies varied for a broad spectrum of T1DM. Here, we systemically profiled auto-antibodies from serum samples of 16 T1DM, 16 type 2 diabetes (T2DM) patients, and 27 healthy controls with normal glucose tolerance (NGT) using protein microarrays containing 9,480 proteins. Among 9,480 different proteins on the array, we identified novel auto-antibody candidates (EEF1A1-AAb and UBE2L3-AAb) by M-test coupled with PLS-DA. These auto-antibodies were highly present in T1DM than controls and detected in 40% of T1DM without GADA. Furthermore, these auto-antibodies might help to differentiate subtype of T1DM when combined with GADA. These novel auto-antibodies provide new diagnostic information of T1DM, as well as new insights into the pathogenesis of T1DM. Auto-antibodies from serum samples were profiled using a high-density, fluorescence-based protein microarray containing duplicate spots of 9,480 human proteins derived from the Ultimate ORF collection The cohort of patients and controls consisted of 16 T1DM, 16 T2DM patients, and 27 healthy controls with NGT. This cohort was used to screen candidate auto-antibodies using protein microarrays (ProtoArray platform version 5.0, Invitrogen Corp., Carlsbad, CA). Serum samples were drawn from T1DM patients who have 1) fasting C-peptide level <0.3 nmol/L or serum C-peptide <0.6 nmol/L after glucagon loading, 2) initiation of insulin treatment within six months after diagnosis, and 3) duration of diabetes M-bM-^IM-$12 months. Mean age of T1DM in the first cohort was 42 M-BM-1 16 years. The control serum samples were obtained from T2DM patients who were treated only with oral anti-diabetic drug at least 5 years and from NGTs who had no history of diabetes, no first-degree relatives with diabetes, a fasting plasma glucose concentration of <6.1 mmol/l, and a HbA1c value of <5.8%.
2014-07-30 | E-GEOD-50866 | biostudies-arrayexpress
Project description:Polyubiquitination analysis upon GSK3 inhibition in HeLa cell extracts
| PRJNA218117 | ENA
Project description:High-density lipoproteins (HDLs) protect pancreatic β cells against apoptosis. This property might be related to the increased risk to develop diabetes in patients with low HDL blood levels. However, the mechanisms by which HDLs protect β cells are poorly characterized. Here we use a transcriptomic approach to identify genes differentially modulated by HDLs in β cells subjected to apoptotic stimuli. Experiment Overall Design: Tested conditions on beta-TC3 cells for 6h (in triplicates for each condition; GSK3 and GSK4 indicate two independent HDL preparations): Experiment Overall Design: Complete Medium + Vehicle [V] Experiment Overall Design: Complete Medium + HDL (prep. HDL GSK3 or HDL GSK4) [HDL] Experiment Overall Design: Starvation medium + Vehicle [V.S] Experiment Overall Design: Starvation medium + HDL (prep. HDL GSK3 or HDL GSK4) [HDL.S]
2009-08-22 | E-GEOD-17647 | biostudies-arrayexpress
Project description:Numerous leucine-rich repeat kinase 2 mutations identified throughout the protein are associated with Parkinson disease, however the activating G2019S kinase domain mutation is currently regarded as the most common cause of familial and sporadic forms of this disorder. Despite studies demonstrating the prominent role that its kinase activity plays in the pathobiology of leucine-rich repeat kinase 2, few substrates have been identified and only a subset of these have been linked to disease. Therefore, we utilized protein microarrays to screen over 9,000 human proteins in an unbiased radiometric assay for potential targets of the kinase. ProtoArrayM-bM-^DM-" Human Protein Microarrays v5.0 (Invitrogen, Carlsbad, CA, USA) were used following the manufactureM-bM-^@M-^Ys protocol (ProtoArray Kinase Substrate Identification Kit). Briefly, slides were equilibrated at 4C for 15 min before blocking in 1% BSA in PBS for 1 h at 4oC with gentle shaking. Recombinant G2019S or D1994A glutathione-S-transferase (GST)-LRRK2 (970-2527) (Invitrogen) was diluted to 50nM in 20mM Tris (pH 7.5), 10mM MgCl2, 1mM EGTA, 1mM Na3VO4, 5mM beta-glycerophosphate, 2mM DTT, 0.02% polysorbate 20, and 10 mCi /mL of [gamma- 33P]ATP (33 nM final concentration) in a total volume of 120uL. Slides were overlayed with buffer alone, or buffer containing G2019S or D1994A LRRK2, then covered with a coverslip and placed in a 50 mL conical tube for 1 h at 30oC. Afterwards, slides were washed with 0.5% SDS buffer and water followed by centrifugation. Dried slides were exposed to a PhosphorImager plate (Amersham Biosciences, Piscataway, NJ, USA), and scanned on a Storm 840 (Molecular Dynamics, Inc., Sunnyvale, CA, USA) at 50 microns.
2012-07-13 | E-GEOD-30496 | biostudies-arrayexpress
Project description:Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway. For microarray screening, Invitrogen Protoarray v4.0 protein microarrays were used. Human NEMO expressed as a C-terminal GST fusion was purified from E. coli lysates and labelled with biotin. NEMO or biotinylated GST were applied to the microarrays and binding partners detected using streptavidin-Alexa Fluor 647. Significant interactors on both arrays were detected using Invitrogen Protoarray Prospector software and a Z-score cutoff of 3.0. Following subtraction of interactors present on the GST control array, a final set of significant NEMO interactors was derived. Full experimental details are supplied in Fenner, B. J., Scannell, M. & Prehn, J. H. M. (2010). Expanding the substantial interactome of NEMO using protein microarrays. PLoS ONE (in press).
2010-06-19 | E-GEOD-19841 | biostudies-arrayexpress
Project description:The aim of the project was to obtain murine hepatoma MH22a [1] cell subline, resistant to anticancer agent 3-amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ), and examine the changes of the protein expression as compared with parental cells, with the main emphasis on the changes of redox proteins. MH22a cells obtained from the Institute of Cytology of the Russian Academy of Sciences (St. Petersburg, Russia), were grown at 37°C in DMEM medium, supplemented with 10% fetal bovine serum and antibiotics in T25 flasks until reaching 70-80% confluence. The medium is then changed to an analogous medium with 5.0 μM TPZ, and, depending on cell viability, 50% of the medium with TPZ is changed 1-2 times a week, repeating that procedure for 3.5 months to form a stable monolayer. The resulting monolayer is inoculated 1:2 and further grown by changing 50% of the culture medium twice a week until re-inoculation. 20 passages were made over 2.5 months in total. We were unable to obtain a cell subline in a stepwise fashion by growing the cells in increasing concentrations of TPZ, because its concentration increase up to 10 μM caused cell death after 1 month. For analysis, one flask per each sample, 3 samples of parental cell line and 3 samples of TPZ-resistant cell line (between 15th and 20th passages), were selected. Cells were rinsed three times with PBS, detached with TrypLE Express (ThermoFisher Scientific, Waltham, MA, USA) and cell concentration and viability was determined with Trypan blue and Countess 2 (ThermoFisher Scientific, Waltham, MA, USA) automated cell counter. Cells were rinsed by centrifuging 500xg for 5 min and suspending in PBS three times. Finally, cells were flash frozen in liquid nitrogen and transferred to -86°C freezer for storage. [1] European Collection of Authenticated Cell Cultures (ECACC), Catalogue No. 96121721.
2023-05-10 | PXD039876 | Pride
Project description:Microarray technology has evolved as a powerful tool over the last decade, to identify biomarkers and study the mechanisms of diseases. We propose a novel application of integrated genomics by combining transcriptional levels with serological antibody profiling after kidney transplantation, with the aim of uncovering the relative immunogenicity of seven different renal compartments after allo-transplantation. Thirty-six paired pre- and post-transplant serum samples were examined from eighteen transplant recipients, across 5,056 protein targets on the ProtoArray V3.0 platform. Normal renal compartment-specific gene expression data from a cDNA platform were re-analyzed and both the cDNA and the ProtoArray platforms were re-annotated to most up-to-date NCBI gene identifiers; 3,835 genes/proteins are measured on both platforms. Antibody levels were ranked for individual patients and the hypergeometric enrichment statistic was applied on mapped compartment-specific expression data. We discovered that after transplantation, in addition to HLA and MICA responses, temporal alloimmune responses are seen against non-HLA antigens specific to different compartments of the kidney, with highest level responses noted against renal pelvis and cortex specific antigens. The renal medulla is of low immunogenicity as none of the outer or inner medulla specific targets generated significant post-transplant antibody responses. Immunohistochemistry confirmed pelvis and cortex specific localizations of selected targeted antigens, supporting the robust nature of this discovery. This study provides a road map of renal compartment-specific non-HLA antigenic targets responsible for generating alloimmune responses, opening the door for clinical correlations with post-transplant dysfunctional states to be determined. Keywords: alloimmune response after kidney transplantation Plasma profiling using Protein Microarray: Serum antibodies were profiled using Invitrogen ProtoArray® Human Protein Microarray v3.0 technology (Invitrogen, Carlsbad, CA). This platform contains 5,056 non-redundant human proteins expressed in a baculovirus system, purified from insect cells and printed in duplicate onto a nitrocellulose-coated glass slide. Five mL serum diluted in PBST buffer at 1:150 was applied for 90 minutes onto the Protoarray, after blocking with blocking buffer for 1 hour. The slides were then washed with 5ml fresh PBST buffer, 4 times for 10 minutes each, and probed with secondary antibody (goat anti-human Alexa 647, Molecular Probes, Eugene, OR) for 90 minutes. Finally, after a second washing with PBST buffer, the slides were dried and scanned using a fluorescent microarray scanner (GSI Luminoics Perkin-Elmer scanner). All steps were carried out on a rotating platform at 4 ºC. ProtoArray data acquisition and measurement: The slides were scanned at a PMT gain of 60% with a laser power of 90% and a focus point of 0 μm. Fluorescence intensity data were acquired using GenePix Pro 6.0 software (Molecular devices, Sunnyvale, CA) with the appropriate â.galâ file downloaded from the ProtoArray central portal on the Invitrogen website (http://www.invitrogen.com/protoarray) by submitting the barcode of each ProtoArray slide.
2010-05-26 | E-GEOD-10452 | biostudies-arrayexpress
Project description:Identifying the targets of immune response after allogeneic hematopoietic cell transplantation (HCT) promises to provide relevant immune therapy candidate proteins. We used protein microarrays to serologically identify Nucleolar and Spindle Associated Protein 1 (NuSAP1) and Chromatin Assembly Factor 1, subunit B (p60) [CHAF1b] as targets of new antibody responses that developed after allogeneic HCT. Western blots and ELISA validated their post-HCT recognition and enabled ELISA testing of 120 other allo-HCT patients. CHAF1b specific antibodies were predominantly detected in AML patients whereas NuSAP1 specific antibodies were exclusively detected in AML patients one year post-transplant (p<0.0001). Complete genomic exon sequencing failed to identify a nonsynonymous SNP for NuSAP1 and CHAF1b between the donor and recipient cells. Expression profiles and RT-PCR showed NuSAP1 was predominately expressed in the bone marrow CD34+CD90+ hematopoietic stem cells (HSC), leukemic cell lines and B lymphoblasts as compared to other tissues or cells. Thus, NuSAP1 is recognized as an immunogenic antigen in 65% AML patients and suggests a tumor antigen role. In conclusion, clinically important tumor antigens can be identified as new antibody targets after allogeneic HCT using high density protein microarrays Plasma Profiling using ProtoArrays: Protein microarrays were obtained (ProtoArrays⢠V3, Invitrogen Corporation, Carlsbad, CA) displaying 5,056 full-length human proteins, derived from the Ultimate ORF collection (Invitrogen Corp., Carlsbad, CA) printed in duplicate with N-terminal GST epitopes expressed in Baculovirus and affinity purified under native conditions maintaining their cellular enzymatic activities/native conformations. The arrays also contained control spots consisting of buffer, empty spots, and human IgG printed in four different concentrations (IgG[1] to IgG[4]). The arrays were incubated with blocking buffer for one hr followed by application of the plasma sample diluted 1:150 for 90 min. After washing the array four times for 10 min each with PBST buffer, secondary antibody was added (goat anti-human Alexa 647, Molecular Probes, Eugene, OR) for 90 min. After washing the slides with PBST buffer, the arrays were dried and fluorescent intensity was detected using a microarray scanner (GenePix 4000B, Molecular Devices, CA). All incubations were carried out on a rotating platform at 4 ºC. ProtoArray data acquisition and measurement: The slides were scanned at a PMT gain of 60% with a laser power of 90% and a focus point of 0 μm. Fluorescence intensity data were acquired using GenePix Pro 6.0 software (Molecular devices, Sunnyvale, CA) with the appropriate â.galâ file downloaded from the ProtoArray central portal on the Invitrogen website (http://www.invitrogen.com/protoarray) by submitting the barcode of each ProtoArray slide
2010-06-21 | E-GEOD-15255 | biostudies-arrayexpress
Project description:Using this approach the dasatinib-oligonucleotide conjugates was applied to planar arrays of >9,000 human proteins spotted in two technical replicates. All proteins in the commercially available ProtoArray® Human Protein MicroArray (Thermo Fisher Scientific) have been purified and arrayed under native conditions to allow such studies. We adopted this format for investigation of the binding profiles of the drug-oligonucleotide conjugates. Fluorophore-labeled drugs have previously been used to measure binding in protein arrays. The oligonucleotide-conjugated constructed allowed for locally amplified detection via RCA. Circularizing oligonucleotides (padlock probes) were designed with 5’ and 3’ ends complementary to adjacent segments of the oligonucleotides conjugated to the drug molecules. Once converted to oligonucleotide circles by ligation, the probes were replicated through localized RCA, primed by the drug-conjugated oligonucleotides, and visualized using fluorescence-labeled hybridization probes to the repeated sequence of the RCA products. RCA offers a signal enhancement of several hundredfold over singly fluorophore labeled compounds, permitting visualization of even single bound drug probes
2022-09-12 | E-MTAB-12192 | biostudies-arrayexpress