Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/sample_metadata.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/Pulldown-proteinGroups.renamed.3br.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/CoIP-proteinGroups.renames.3br.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/Ctrl_cFos_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/GnRH_cFos_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/GnRH_Egr1_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/Ctrl_FSH_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/GnRH_LH_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/GnRH_FSH_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/Ctrl_LH_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/Ctrl_cFos_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/Ctrl_Egr1_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/GnRH_FSH_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/GnRH_Egr1_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/GnRH_Egr1_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/Ctrl_cFos_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/GnRH_LH_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/Ctrl_Egr1_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/GnRH_cFos_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/Ctrl_LH_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/Ctrl_FSH_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/GnRH_FSH_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/GnRH_LH_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/GnRH_cFos_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/Ctrl_Egr1_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/Ctrl_FSH_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075636/Ctrl_LH_1.rawprimaryOK200Djurdjica.Coss@medsch.ucr.eduBrandon LeMass SpectrometryBottom-up proteomicsGonadotropinshttps://www.ebi.ac.uk/pride/archive/projects/PXD075636GonadtrophCell CultureFor DNA pulldown assays, 5’ Biotinylated sense and anti-sense DNA oligos containing LHβ and FSHβ promoter sequences were designed to include GnRH response elements with Egr1 and AP1 binding sequences, respectively (Table 1, Integrated DNA Technologies). Lyophilized oligos were resuspended in TE buffer at a final concentration of 100 uM. Oligos were annealed by combining an equal molar amount of sense and anti-sense strands for LHβ and FSHβ separately. For the DNA pulldown assay, a pre-clearing reaction was performed using 500ug of nuclear extract, 25ug Poly dIdC and streptavidin agarose beads, in the binding buffer (100mM HEPES pH 7.8, 250mM KCl, 10mM MgCl2, 0.1% NP-40, 0.1M DTT) for 1 hour at 4oC. Pre-clearing reaction was centrifuged at 6000 RPM for 5 minutes at 4oC and supernatant was transferred to a new tube with 10pmol of double stranded biotinylated LHβ or FSHβ oligo and incubated on a rotator for 3 hours at 4oC. After binding, streptavidin agarose beads were added to the reaction and incubated overnight at 4oC. The following day, the samples were precipitated at 6000 RPM for 5 minutes at 4oC and supernatant was discarded. Streptavidin agarose beads were washed 5 times for 5 minutes at 4oC in wash buffer (100mM HEPES pH 7.9, 500mM KCl, 10mM MgCl2, 0.1% NP-40 in water) and centrifuged at 6000 RPM at 5 minutes at RT. After last wash, beads were resuspended in 50uL of 2X Laemmli buffer (125mM Tris-HCl pH 6.8, 2% (w/v) SDS, 20% (v/v) glycerol and 355mM 2mercaptoethanol) and boiled for 15 minutes at 95oC. Binding complexes eluted in 2X Laemmli buffer were loaded on a 10% acrylamide SDS-PAGE gel along with protein size markers (Precision Plus Protein Standards, Bio-Rad, Hercules, CA, USA). Proteins were separated by electrophoresis and stained with Coomassie blue stain for 1 hour at RT (Bio-Rad, Hercules, CA, USA). Lanes containing samples were excised and de-stained in 10% acetic acid and 20% methanol solution. For co-immunoprecipitation, Nuclear extracts from either vehicle or GnRH-treated LβT2 cells were isolated using cell fractionation as described above. 1mg of nuclear extract from vehicle-treated or GnRH-treated nuclear extracts was incubated on ice with either 2.5ug of rabbit anti-mouse cFOS (Santa Cruz Biotechnology, Cat no sc-253, Clone K-25) or 2.5ug of rabbit anti-mouse Egr1 (Santa Cruz Biotechnology, Cat no sc-110, Clone 588) and rotated for 3 hours at 4oC. After incubation, complexes were incubated with Protein A/G agarose beads for 1 hour at 4oC while rotating. Complexes were then spun down at 4000xg for 5 minutes at 4oC and washed 5 times for 5 minutes each with wash buffer (20mM Tris pH 7.4, 140mM NaCl, 0.05mM EDTA pH 8.0, 1mM EGTA pH 8.0, 0.1% NP-40, 1X Sigma Protease Inhibitor Cocktail, 1mM PMSF in water) and centrifuged at 4000xg for 5 minutes at 4oC after each wash. Complexes eluted in 2X Laemmli buffer were loaded on a 10% acrylamide SDS-PAGE gel, stained with Coomassie blue stain and de-stained in 10% acetic acid and 20% methanol solution. Gel slices from both assays were sent to the UCR IIGB Proteomics core facility for in-gel tryptic digestion and LC-MS on a Thermo Scientific Orbitrap Fusion.PrideNot availableThe raw data were processed and analyzed using MaxQuant (version 2.1.4.0) with uniprot "Mouse_UP000000589_10090" database. In particular, mass tolerances for precursor and fragment ions were 6 and 10 ppm, respectively, the minimum peptide length was 7 amino acids, and the maximum number of missed cleavages for trypsin was 2.ProteomicsDjurdjica CossPARTIALDivision of Biomedical Sciences University of California, Riverside Riverside, CA 92521Mus Musculus (mouse)42343567 Ruggiero-Ruff RE, Le BH, Coss D. Discovery proteomics identification of factors contributing to gonadotropin β expression. Endocrinology. 2026:bqag073 10.1210/endocr/bqag073brandon.le@ucr.eduUniversity of California Riverside Institute for Integrative Genome BiologyUnited StatesLuteinizing hormone (LH) and follicle-stimulating hormone (FSH), critical for reproduction, are heterodimers of a common α subunit and unique β subunits, that are limiting components in the mature hormone synthesis. GnRH from the hypothalamus regulates synthesis of LH and FSH β-subunits. Previous studies identified signaling pathways and transcription factors that mediate GnRH induction of β-subunits. However, these studies do not fully explain gonadotrope responsiveness to GnRH or mechanisms of how a single hormone can regulate two β-subunits differentially. We postulated additional transcription factors, coactivators or corepressors, that are awaiting identification. To provide novel candidates, we used discovery proteomics of protein complexes that associate with gonadotropin β promoters regions that contain GnRH-responsive elements, and protein complexes that interact with transcription factors that are activated by GnRH to induce gonadotropin β genes. Data are available via ProteomeXchange with identifier PXD075636. Using computational tools, such as Intervene and Tidyproteomics, we compare and contrast proteins in complexes to identify coactivators or corepressors that may be unique or in common for both gonadotropin β subunit transcriptional upregulation or repression, novel transcription factors, and proteins that may regulate both gonadotropin β gene expression, proteins specific to each promoter, and proteins that are recruited via interaction with intermediate GnRH-induced transcription factors. Our studies can serve as a resource to interrogate potential candidates in cell cultures and in vivo for their roles in gonadotropin β expression. Understanding the mechanisms whereby GnRH regulates gonadotropin hormone levels will provide insight into the physiology and pathophysiology of the reproductive system.Discovery proteomics identification of factors contributing to gonadotropin β expression.Ruggiero-Ruff Rebecca E RE, Le Brandon H BH, Coss Djurdjica DfalseIdentification of protein complexes in Gonadotropin signaling using discovery proteomicsWe utilized discovery proteomics to identify putative factors that are recruited to the GnRH-responsive promoter regions of gonadotropin β genes (Egr1 and cFos). We also used this approach to identify proteins that associate with immediate early genes that regulate LHβ and FSHβ.2026-06-292026-03-14PXD075636NEWT:1773NEWT:3555NEWT:1182590NEWT:10090NEWT:749200NEWT:35554NEWT:4120NEWT:5693NEWT:347515NEWT:1216979NEWT:307972NEWT:92867NEWT:990346NEWT:544496NEWT:5334NEWT:145953NEWT:257309NEWT:284812NEWT:115104NEWT:43330NEWT:67825NEWT:44544NEWT:13076NEWT:544404NEWT:3702NEWT:8839NEWT:4232NEWT:1736309NEWT:4113NEWT:7227NEWT:11298NEWT:885318NEWT:4081NEWT:876138NEWT:554NEWT:5691NEWT:260710NEWT:106592NEWT:237561NEWT:9913NEWT:10036NEWT:4100NEWT:7574NEWT:1351NEWT:1076NEWT:6763NEWT:7215NEWT:8030NEWT:380394NEWT:272563NEWT:507601NEWT:1639NEWT:188229NCBITaxon:79857NEWT:746360NEWT:6239NEWT:135588NEWT:135622NEWT:6915NEWT:9986NEWT:101510NEWT:95486NEWT:3880NEWT:58002NEWT:9103NEWT:4577NEWT:146479NEWT:1000589NEWT:145943NEWT:85962NEWT:160488NEWT:317447NEWT:3635NEWT:7955NCBITaxon:2NEWT:235443NEWT:985076NEWT:7959NEWT:2261NEWT:3197NEWT:9615NEWT:884019NEWT:4565NEWT:1264690NEWT:169963NCBITaxon:38727NEWT:36329NEWT:34305NEWT:59729NCBITaxon:183674NEWT:626528NEWT:139927NEWT:4558NEWT:9606NEWT:367830NEWT:243230NEWT:931281NEWT:7029NEWT:1283300NEWT:334747NEWT:470NCBITaxon:79824NCBITaxon:4563NEWT:3218NEWT:5759NEWT:9838NCBITaxon:9615NEWT:1736231NEWT:1193501NEWT:6287NEWT:6326NEWT:9796NEWT:2762NEWT:5476NEWT:562NEWT:260707NEWT:287NEWT:10117NEWT:10116NEWT:1280NEWT:1836NEWT:29760NEWT:260705NEWT:1148NEWT:4932NEWT:70448NEWT:9825NEWT:3603NEWT:698936NEWT:39946NEWT:11676NEWT:9823NEWT:100226NCBITaxon:6073NEWT:4896NEWT:6279NEWT:7370NEWT:573NEWT:6282NEWT:7091423435670000-0001-8651-2186