Journal: Journal of Mammary Gland Biology and Neoplasia

Article Title: Disease-Associated Mutations of the STAT5B SH2 Domain Regulate Cytokine-Driven Enhancer Function and Mammary Development

doi: 10.1007/s10911-025-09582-8

Figure Lengend Snippet: STAT5B mutations at Y665 in the mouse genome. A Schematic illustration of the STAT5B protein domains. Amino acid locations of the variants are highlighted. B Three-dimensional model of STAT5B protein structure presenting the location of the Y665 residue mutated in the mouse genome. C Sanger sequencing chromatograms showing Y665 (wild-type, WT) and the introduction of SNPs resulting in the two missense mutations, Stat5b Y665 F (Y665F) and Stat5b Y665H (Y665H) mutants. The red shade indicates the altered codons converting Y665 to Y665F and Y665H, respectively. D Number and percentage of offspring from intercrossed heterozygote parents (Y665H, n = 301; Y665F, n = 543). The statistical relationship between the percentage and the expected Mendelian ratios was assessed using a Chi-square test. E Dot plot presenting the weight of individual mice in each group from 6, 7, 8 and 9 weeks of age. Wild-type mice are from litters weaned from heterozygous Stat5b Y665H (gray) and Stat5b Y665F (black) dams. Results are presented as the means ± SEM of independent biological replicates (Y665, n = 13; Y665H, n = 6; Y665F, n = 17). A two-way ANOVA followed by Tukey’s multiple comparisons test was used to evaluate the statistical significance of differences between groups. ** p < 0.001, **** p < 0.0001

Article Snippet: The expression of STAT5A and STAT5B proteins was detected by overnight incubation with primary antibodies: STAT5A (1:200, Santa Cruz Biotechnology, sc-271542) and STAT5B (1:200, R&D Systems, AF1584) at room temperature.

Techniques: Residue, Sequencing

Journal: Journal of Mammary Gland Biology and Neoplasia

Article Title: Disease-Associated Mutations of the STAT5B SH2 Domain Regulate Cytokine-Driven Enhancer Function and Mammary Development

doi: 10.1007/s10911-025-09582-8

Figure Lengend Snippet: Impaired alveolar development in STAT5B Y665 mutant mice at day 18.5 of pregnancy. A Sections of mammary tissue stained with hematoxylin and eosin at day 18.5 of pregnancy with high magnification (x400, Scale bars, 100 μm). The arrow and Asterix indicates alveoli and lumen. Y665, n = 3; Y665H, n = 3; Y665F, n = 4. 50–100 of the alveolar architecture on each slide was quantitatively analyzed using the ImageJ software, with the results visualized through dot plots. (a) milk-secreting alveoli; (b) alveolar lumen; (c) alveolar epithelial cells. B , D and E Heatmaps showing fold activity of significantly regulated genes of milk proteins in red, enzymes in green, fat metabolism in gray, membrane transporters in blue, transcription factors in black (B), cell marker genes (D) and skin-related genes (E) in STAT5B Y665H and STAT5B Y665F mutants compared to WT (RNA-seq; Y665, n = 3; Y665H, n = 3; Y665F, n = 4). C Dot plots with the normalized read counts from RNA-seq to mRNA levels of Stat5 genes controlled by autoregulatory enhancer. Results are shown as the means ± SEM of independent biological replicates (Y665, n = 3; Y665H, n = 3). The Benjamini-Hochberg adjusted p -value was used for significance. *** p < 0.0001, **** p < 0.0001

Article Snippet: The expression of STAT5A and STAT5B proteins was detected by overnight incubation with primary antibodies: STAT5A (1:200, Santa Cruz Biotechnology, sc-271542) and STAT5B (1:200, R&D Systems, AF1584) at room temperature.

Techniques: Mutagenesis, Staining, Software, Activity Assay, Membrane, Marker, RNA Sequencing

Journal: Journal of Mammary Gland Biology and Neoplasia

Article Title: Disease-Associated Mutations of the STAT5B SH2 Domain Regulate Cytokine-Driven Enhancer Function and Mammary Development

doi: 10.1007/s10911-025-09582-8

Figure Lengend Snippet: Nuclear localization of STAT5A and STAT5B in STAT5B Y665 mutant mice. Staining of STAT5A and STAT5B proteins at day 18.5 of the first pregnancy with high magnification (x10 and x400, Scale bars, 100 μm). The arrow indicates alveolar lumen ( a ), alveolar epithelial cells ( b ), fibroblasts ( c ) and ( d ) fat

Article Snippet: The expression of STAT5A and STAT5B proteins was detected by overnight incubation with primary antibodies: STAT5A (1:200, Santa Cruz Biotechnology, sc-271542) and STAT5B (1:200, R&D Systems, AF1584) at room temperature.

Techniques: Mutagenesis, Staining

Journal: Journal of Mammary Gland Biology and Neoplasia

Article Title: Disease-Associated Mutations of the STAT5B SH2 Domain Regulate Cytokine-Driven Enhancer Function and Mammary Development

doi: 10.1007/s10911-025-09582-8

Figure Lengend Snippet: Genomic features of the genes regulated by STAT5B Y665 mutations. A Coverage plots displaying the patterns of H3K27ac marks and STAT5B and STAT5A binding on 440 mammary-specific super-enhancers at day 18.5 of pregnancy. Black, Y665; blue, Y665H; red, Y665F. B Binding of STAT5B and NFIB, H3K27ac, and Pol II at gene loci of milk protein genes controlled by mammary-specific super-enhancers, transporter genes, and skin genes in mammary tissue of wild type (Y665) and Stat5b Y665H (Y665H) mice at day 18.5 of pregnancy. C STAT5B and NFIB binding, H3K27ac and Pol II loading at gene loci of milk protein genes controlled by mammary-specific super-enhancers, transporter gene and skin gene in mammary tissue of Y665 and Stat5b Y665F (Y665F) mice. Y665, n = 2; Y665H, n = 2; Y665F, n = 2

Article Snippet: The expression of STAT5A and STAT5B proteins was detected by overnight incubation with primary antibodies: STAT5A (1:200, Santa Cruz Biotechnology, sc-271542) and STAT5B (1:200, R&D Systems, AF1584) at room temperature.

Techniques: Binding Assay

Journal: Journal of Mammary Gland Biology and Neoplasia

Article Title: Disease-Associated Mutations of the STAT5B SH2 Domain Regulate Cytokine-Driven Enhancer Function and Mammary Development

doi: 10.1007/s10911-025-09582-8

Figure Lengend Snippet: Alveolar development of STAT5B Y665F mutants at mid-pregnancy. A Sections of mammary tissue stained with hematoxylin and eosin at day 13.5 of pregnancy with high magnification (x400, Scale bars, 100 μm). The arrow indicates alveoli. Y665, n = 3; Y665F, n = 4. The alveolar architecture was quantitatively analyzed using the ImageJ software, with the results visualized through dot plots. (a) milk-secreting alveoli; (b) alveolar lumen; (c) alveolar epithelial cells. B Bar graphs showing fold activity of genes of milk proteins, transporters and transcription factors in STAT5B Y665F (Y665F) mice compared to wild type (Y665) (RAN-seq; Y665, n = 3; Y665F, n = 4). C Heatmaps presenting fold activity of milk protein genes in red, transporter genes in blue, transcription factors in black (left) and skin genes (right) in STAT5B Y665F mice at day 13.5 of pregnancy (presented as p13) compared to WT at day 18.5 of pregnancy (presented as p18) (RAN-seq; Y665 at p18, n = 3; Y665F at p13, n = 4). D Binding of STAT5B and NFIB, H3K27ac, and Pol II at gene loci of milk protein genes controlled by mammary-specific super-enhancers in mammary tissue of Y665 and Y665F mice at day 13.5 of pregnancy. Y665, n = 2; Y665F, n = 2

Article Snippet: The expression of STAT5A and STAT5B proteins was detected by overnight incubation with primary antibodies: STAT5A (1:200, Santa Cruz Biotechnology, sc-271542) and STAT5B (1:200, R&D Systems, AF1584) at room temperature.

Techniques: Staining, Software, Activity Assay, Binding Assay

Journal: Journal of Mammary Gland Biology and Neoplasia

Article Title: Disease-Associated Mutations of the STAT5B SH2 Domain Regulate Cytokine-Driven Enhancer Function and Mammary Development

doi: 10.1007/s10911-025-09582-8

Figure Lengend Snippet: Partial activation of mammary function and lactation in STAT5B Y665H mice after the second pregnancy. A Body weight of pups at day ten of lactation (L10) after the second pregnancy of homozygous Stat5b Y665H mice and after the first pregnancy of heterozygous Stat5b Y665H mice. The weight of pups from 3–4 females was measured and normalized by the number of pups. A t -test was utilized to evaluate the statistical significance between pups from homozygous females at second pregnancy and heterozygous females at first pregnancy. Homozygous Stat5b Y665H mice, n = 4 cages; heterozygous Stat5b Y665H mice, n = 3 cages. * p < 0.05. B Expression of representative mammary genes was measured in mammary tissue of Y665 (WT) mice collected at day 10 of lactation (L10), and of STAT5B Y665H mice at L10 after the second pregnancy by RNA-seq (Y665, n = 3; Y665H, n = 4). The p18 data (Fig. ) is presented alongside the L10 data to highlight the relative reduction in gene expression between p18 and L10 in Y665H mice. C Binding of STAT5A and STAT5B, H3K27ac, and Pol II at Stat5 and Wap super-enhancer loci controlled by prolactin in mammary tissue of Y665 and Y665H mice at day 10 of lactation. The Socs2 locus was presented as a ChIP-seq control. Y665, n = 2; Y665H, n = 2. D Sections of mammary tissue stained with hematoxylin and eosin at day 10 of lactation after the second pregnancy with high magnification (x400, Scale bars, 100 μm). The arrow and Asterix indicates alveoli and lumen. Y665, n = 1; Y665H, n = 3. The alveolar architecture was quantitatively analyzed using the ImageJ software, with the results visualized through dot plots. (a) milk-secreting alveoli; (b) alveolar lumen; (c) alveolar epithelial cells

Article Snippet: The expression of STAT5A and STAT5B proteins was detected by overnight incubation with primary antibodies: STAT5A (1:200, Santa Cruz Biotechnology, sc-271542) and STAT5B (1:200, R&D Systems, AF1584) at room temperature.

Techniques: Activation Assay, Expressing, RNA Sequencing, Gene Expression, Binding Assay, ChIP-sequencing, Control, Staining, Software

Journal: Journal of Mammary Gland Biology and Neoplasia

Article Title: Disease-Associated Mutations of the STAT5B SH2 Domain Regulate Cytokine-Driven Enhancer Function and Mammary Development

doi: 10.1007/s10911-025-09582-8

Figure Lengend Snippet: Regulation of key fat metabolic genes. A-B Dot plots with the normalized read counts from RNA-seq to mRNA levels of Olah and Fabp3 genes at virgin and L10 of wild type mice (A) and at p18 of wild type and Y665 mutant mice (B). Results are shown as the means ± SEM of independent biological replicates (WT at virgin, n = 3; WT at L10, n = 4; Y665 at p18, n = 3; Y665H at p18, n = 3). p -values are from 2-way ANOVA followed by Sidak’s multiple comparisons test between WT and mutants. ** p < 0.001, **** p < 0.0001. C Binding of SAT5A and STAT5B, H3K27ac, and Pol II at Olah and Fabp3 gene loci controlled by prolactin in mammary tissue of Y665 and Y665H mice at day 10 of lactation. Y665, n = 2; Y665H, n = 2

Article Snippet: The expression of STAT5A and STAT5B proteins was detected by overnight incubation with primary antibodies: STAT5A (1:200, Santa Cruz Biotechnology, sc-271542) and STAT5B (1:200, R&D Systems, AF1584) at room temperature.

Techniques: RNA Sequencing, Mutagenesis, Binding Assay

Journal: Journal of Mammary Gland Biology and Neoplasia

Article Title: Disease-Associated Mutations of the STAT5B SH2 Domain Regulate Cytokine-Driven Enhancer Function and Mammary Development

doi: 10.1007/s10911-025-09582-8

Figure Lengend Snippet: Dimerization of STAT5A and STAT5B protein. A Strategy of co-ChIP-seq. STAT5A bound chromatin from mammary tissue of WT mice at day 1 of lactation (L1) was precipitated using STAT5A antibody and eluted from Ig beads. Secondly, STAT5B bound chromatin was precipitated using STAT5B and eluted from the beads. STAT5A motifs from 1st ChIP and STAT5B motifs from 2nd ChIP were identified by sequencing. B Co-ChIP profile at the representative milk protein Wap gene and common STAT target Socs2 gene. C STAT5A and STAT5B binding at gene loci of milk protein Wap gene and common STAT target Socs2 gene in the mammary gland of Y665 (WT) and Y665H mice at day 18.5 of pregnancy (p18). D STAT5A and STAT5B binding in the mammary gland of Y665 (WT) and Y665F mice at day 13.5 of pregnancy (p13)

Article Snippet: The expression of STAT5A and STAT5B proteins was detected by overnight incubation with primary antibodies: STAT5A (1:200, Santa Cruz Biotechnology, sc-271542) and STAT5B (1:200, R&D Systems, AF1584) at room temperature.

Techniques: ChIP-sequencing, Sequencing, Binding Assay

Journal: Scientific Reports

Article Title: E3 ubiquitin ligases LNX1 and LNX2 are major regulators of the presynaptic glycine transporter GlyT2

doi: 10.1038/s41598-019-51301-x

Figure Lengend Snippet: Schematic diagram of the modular domain structure of LNX1/2 proteins and sequence alignment of GlyT2 C-terminal region from different species. ( A ) Graphic scheme showing the two main isoforms of LNX1, p70 (short isoform) and p80 (long isoform with RING domain) and LNX2. A previous unbiased screening effort by Guo et al . identified the last 8 amino acids of GlyT2 (DLELGTQC) as a possible interacting peptide for the PDZ II domain of LNX, which recognizes the consensus sequence –[S/T]-X-C*, where * shows the end of the protein. ( B ) Multiple sequence alignment of rat GlyT2 C-terminal region 791–799 from different species. Identical conserved PDZ binding motif (PBM) from different species are shown in bold.

Article Snippet: FLAG-tagged LNX1-p80 was a generous gift from Prof. Jane McGlade (University of Toronto), pClneoMyc mouse LNX1-p70 and pClneoMyc mouse LNX2 were a gift from Yutaka Hata (Addgene plasmids #37009 and #37010, respectively) and pRK5-HA-Ubiquitin-WT was a gift from Ted Dawson (Addgene plasmid #17608).

Techniques: Sequencing, Binding Assay

Journal: Scientific Reports

Article Title: E3 ubiquitin ligases LNX1 and LNX2 are major regulators of the presynaptic glycine transporter GlyT2

doi: 10.1038/s41598-019-51301-x

Figure Lengend Snippet: Identification and characterization of LNX2 as interacting partner of GlyT2. ( A ) COS7 cells expressing GlyT2 and Myc-LNX2 were lysed and subjected to immunoprecipitation against GlyT2 and Myc. Immunoprecipitates were analyzed by immunoblotting for indicated proteins, showing that GlyT2 coimmunoprecipitates LNX2 (middle lane). Correspondingly, LNX2 coimmunoprecipitate GlyT2 (right lane). ( B ) COS7 cells were transiently transfected with GlyT2 and with or without Myc-LNX2. GlyT2 was immunoprecipitated and ubiquitination of the transporter was assayed by immunoblotting with anti-ubiquitin antibody. Blots were probed against GlyT2 to normalize ubiquitination signal against the amount of GlyT2 immunoprecipitated in each case to correct for GlyT2 protein expression. ( C ) Quantification of GlyT2 ubiquitination normalized to the control (no transfection of Myc-LNX2). **p = 0.022, using Kolmogorov-Smirnov test. n(control) = 6, n(LNX2) = 6. ( D ) COS7 cells were transiently transfected with GlyT2 or GlyT2-4KR in combination with pcDNA3 or Myc-LNX2 and GlyT2 expression was measured by immunoblotting. Quantification of the effect of LNX2 on GlyT2 and GlyT2-4KR expression was normalized against tubulin. ( E ) Quantification is shown normalized to the corrected signal in the control in each case with no LNX2 co-transfection (indicated by dashed line). ****p < 1 · 10 −4 , n.s., not significantly different, using Kruskall-Wallis with Dunn’s post hoc test. Number of experiments in each case: n(GlyT2 control) = 8, n(GlyT2-LNX2) = 8, n(GlyT2-4KR) = 4, n(GlyT2-4KR-LNX2) = 4. Note the higher reduction of GlyT2 expression by LNX2 respect to that induced in GlyT2-4KR mutant. Myc immunobloting was used to confirm transfection of LNX2. ( F ) COS7 cells were transiently transfected with GlyT2 in combination with pcDNA3 or Myc-LNX2 and glycine transport rates were measured using [ 3 H]-Glycine transport assays as described inexperimental procedures. Glycine transport shown is normalized against control conditions. ****p < 1 · 10 −4 , n.s., not significantly different, using Mann-Whitney U test. n(control) = 27, n(LNX2) = 27.

Article Snippet: FLAG-tagged LNX1-p80 was a generous gift from Prof. Jane McGlade (University of Toronto), pClneoMyc mouse LNX1-p70 and pClneoMyc mouse LNX2 were a gift from Yutaka Hata (Addgene plasmids #37009 and #37010, respectively) and pRK5-HA-Ubiquitin-WT was a gift from Ted Dawson (Addgene plasmid #17608).

Techniques: Expressing, Immunoprecipitation, Western Blot, Transfection, Ubiquitin Proteomics, Control, Cotransfection, Mutagenesis, MANN-WHITNEY

Journal: Scientific Reports

Article Title: E3 ubiquitin ligases LNX1 and LNX2 are major regulators of the presynaptic glycine transporter GlyT2

doi: 10.1038/s41598-019-51301-x

Figure Lengend Snippet: Silencing of LNX2 in neurons causes upregulation of endogenous GlyT2 and abolish the PKC-mediated regulation of GlyT2. ( A ) Relative mRNA levels of LNX1-p80 and LNX2 were determined by qPCR using Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as housekeeping gene (arbitrary units). n(kidney) = 3, n(primary brainstem and spinal cord neuron cultures) = 3. ( B ) Neuronal cultures were infected with LNX2 shRNA and scrambled shRNA as control. After 12 DIV total RNA was extracted from cells and quantification of LNX2 mRNA was determined by qPCR. LNX2 mRNA in these conditions decreased by 81.4 ± 11,8%. ****p < 1 · 10 −4 , n.s., not significantly different, using Kolmogorov-Smirnov test. n(control shRNA) = 14, n(LNX2 shRNA) = 14. ( C ) Cultured neurons were infected as in ( B ) and western blot analysis were performed using anti-GlyT2 antibodies. Tubulin was used as a loading control. ( D ) Quantification is shown normalized to the corrected signal in the control (scrambled shRNA) indicated by a dashed line. ****p < 1 · 10 −4 , n.s., not significantly different, using Kolmogorov-Smirnov test. n(control shRNA) = 16, n(LNX2 shRNA) = 16. ( E ) Neuronal cultures infected with LNX2 shRNA or scrambled shRNA were treated with or without (vehicle) 1 µM PMA during 2 h and were subjected to western blot analysis using anti-GlyT2 antibodies. Tubulin was used as protein loading control. Note that ablation of LNX2 impairs the effect of PMA on GlyT2 expression. ( F ) Quantification is shown normalized to the corrected signal in the control (vehicle) indicated by dashed line. **p = 0,005, n.s., not significantly different, using Two-way Anova with Sidak’s post hoc test. Number of experiments in each case: n(control shRNA + Veh) = 7, n(control shRNA + PMA) = 7, n(LNX2 shRNA + Veh) = 6, n(LNX2 shRNA + PMA) = 6. ( G ) In neuronal cultures infected and PMA treated as in E glycine transport rates were measured using [ 3 H]-Glycine transport assays. Glycine transport shown is normalized against control conditions in each case. ****p < 1 · 10 −4 , n.s., not significantly different, using Two-way Anova with Sidak’s post hoc test. Number of experiments in each case: n(control shRNA + Veh) = 6, n(control shRNA + PMA) = 6, n(LNX2 shRNA + Veh) = 5, n(LNX2 shRNA + PMA) = 5.

Article Snippet: FLAG-tagged LNX1-p80 was a generous gift from Prof. Jane McGlade (University of Toronto), pClneoMyc mouse LNX1-p70 and pClneoMyc mouse LNX2 were a gift from Yutaka Hata (Addgene plasmids #37009 and #37010, respectively) and pRK5-HA-Ubiquitin-WT was a gift from Ted Dawson (Addgene plasmid #17608).

Techniques: Infection, shRNA, Control, Cell Culture, Western Blot, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Transcriptional Regulation of Nos2 via STAT5B Binding to Nos2 Gene Promoter Mediates Nitric Oxide Production: Relevance in β-Cell Maintenance.

doi: 10.33594/000000010

Figure Lengend Snippet: Fig. 1. STAT5B is a transcriptional regulator of Nos2. A) Chromosome ideogram of mouse Stat5b and Nos2 on chromosome 11(http://genome.ucsc.edu/). B) Cartoon representation of the putative binding site of STAT5B on Nos2 promoter, as identified from ChampionChiP Transcription Factor Search Portal (SABiosciences). C) Chromatin immunoprecipitation in MIN6 cells identified Nos2 as a direct target of STAT5B. Lane 1 - Input DNA, lane 2 - mouse control IgG, lane 3 - anti-RNA polymerase II antibody, lane 4 - anti-STAT5B antibody, lane 5 - DNA ladder. The image is a representation of experiment done thrice independently. D) Bar graph representation of 2-ΔΔCt of Stat5b and Nos2 transcripts showing downregulation in prolactin-induced Stat5b siRNA treated versus control siRNA treated MIN6 cells, calculated with respect to untreated cells and 18SrRNA as the endogenous control. (n = 3). E) Western blots and F) densitometric analysis of STAT5B and NOS2, showing a downregulation in PRL-induced siRNA treated samples, with actin as the loading control (n = 3). P-value was calculated using Student’s t-test.

Article Snippet: Control siRNA (sc-37007) and Stat5b siRNA (sc-37011) were from Santa Cruz Biotechnology (SCBT), Dallas, TX, USA.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Control, Western Blot

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Transcriptional Regulation of Nos2 via STAT5B Binding to Nos2 Gene Promoter Mediates Nitric Oxide Production: Relevance in β-Cell Maintenance.

doi: 10.33594/000000010

Figure Lengend Snippet: Fig. 4. Impact of Stat5b silencing on SOD2 and H2O2. A) Bar graph representation of increased relative transcript levels (2-

Article Snippet: Control siRNA (sc-37007) and Stat5b siRNA (sc-37011) were from Santa Cruz Biotechnology (SCBT), Dallas, TX, USA.

Techniques: