anti h2bub1  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Resistance of estrogen receptor function to BET bromodomain inhibition is mediated by transcriptional coactivator cooperativity

doi: 10.1101/2024.07.25.605008

Figure Lengend Snippet: a , Schematic of the knockin strategy for BRD4-dTAG. b , Agarose gel electrophoretic analysis of PCR products from MCF7 parental cells and MCF7 BRD4-dTAG knockin cells. c , Immunoblot for BRD4 expression in BRD4-dTAG cells treated with dTAG-7 or dTAG V -1 at indicated concentrations for indicated times. dBET6 was added for 2 hours to show changes in Pol II CTD Ser2-P. d , Immunoblot showing effects of different 2-hour treatments on Pol II and H2Bub1 in BRD4-dTAG cells. e , Percentage of MCF7 BRD4-dTAG cell survival after treatment with or without BET inhibitors and dTAG-7 (250 nM) for 6 days. Data shown as mean ± s.d. from biological triplicates. f , Immunoblot showing reduced ER expression in BRD4-dTAG cells upon overnight treatment with JQ1 and dTAG-7. g,h, qPCR of MYC, Cyclin D1 ( h ) and ESR1-Y537 g, mRNA levels in MCF7 BRD4-dTAG ESR1-Y537S cells upon overnight treatment with BET inhibitors and dTAG-7 (500 nM). Because ESR1-Y537S-IRES-GFP is transcribed as a single transcript, ESR1 -Y537S RNA level was monitored by GFP RNA. All cells were grown in normal full medium containing 10% FBS. Data shown as mean ± s.d. from independent triplicates. p values from two-tailed t-tests are depicted with asterisks.

Article Snippet: ERα (#D8H8, WB), H2Bub1 (#5546), MED26 (#13641), and Rpb1 NTD (#D8L4Y, ChIP) antibodies were from Cell Signaling.

Techniques: Knock-In, Agarose Gel Electrophoresis, Western Blot, Expressing, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: USP22 supports the aggressive behavior of basal-like breast cancer by stimulating cellular respiration

doi: 10.1186/s12964-023-01441-5

Figure Lengend Snippet: Mammary tissue-specific loss of Usp22 impairs the stem cell-like properties of the growing murine mammary gland: A Schematic representation of the two transgenes of the MMTV-cre; Usp22 fl/fl mouse model. B Whole mounts staining of mammary glands showing a significant decrease of mammary duct branching density in MMTV-cre; Usp22 fl/fl mice compared to the control group with representative brightfield pictures (left panel) and the respective quantification of branching density and number of ex vivo cultured mammospheres (right panel). C Mammosphere formation assay mammary epithelial cells from MMTV; Usp22 wt/wt and MMTV; Usp22 fl/fl mice. D Hematoxylin and eosin staining (left panel) and immunohistochemical detection of H2Bub1 (right panel) on mammary gland sections from MMTV-cre; Usp22 fl/fl mice and MMTV-cre mice. E-I Brightfield pictures (white scale bar: 100 µm) ( E ), western blot analysis of USP22 protein ( F ), growth kinetics ( G ), colony formation assay ( H ) and mammosphere formation assay ( I ) in siControl- and siUSP22-treated MCF10A and MCF12A cells. J Gene set enrichment analysis (GSEA) performed on the high-throughput RNA sequencing data of siControl- and siUSP22-treated MCF10A cells (accession number: E-MTAB-8247). Statistics: B (right panel): One-way Anova; C, G (based on the area under the curve = AUC); I: Student t-test. * p <0.05, *** p <0.005. All experiments were performed in at least three biological replicates

Article Snippet: Anti-H2Bub1 antibody (mouse, homemade at 1:50 dilution) or anti-USP22 (Sigma-Aldrich, HPA044980 at 1:100 dilution) was diluted on blocking solution and samples were treated overnight.

Techniques: Staining, Ex Vivo, Cell Culture, Tube Formation Assay, Immunohistochemistry, Western Blot, Colony Assay, High Throughput Screening Assay, RNA Sequencing Assay