histone h2b coding sequence  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: RSRC2 is a novel RNA-binding protein that safeguards mitotic fidelity by interacting with the lncRNA C1QTNF1-AS1

doi: 10.1093/nar/gkag229

Figure Lengend Snippet: Loss of C1QTNF1–AS1 or RSRC2 results in increased mitotic index and intact SAC signalling. ( A ) Representative images of metaphase cells stained for kinetochores (CREST, cyan), SAC (Mad2, green), and DNA (Hoescht, magenta) after LNA-mediated depletion of C1QTNF1–AS1 in HCT116 cells. ( B ) Insets show examples of MAD2 and CREST staining from C1QTNF1–AS1 -depleted cells, as in panel (A); scale bar: 0.5 µm. ( C ) Characterization of SAC activity in HCT116 cells after LNA-mediated depletion of C1QTNF1–AS1 . Percentage of mitotic cells with Mad2 signal on misaligned chromosomes using the same antibodies as in panel (A); N = 3 ( n (Ctl A) =67; n ( C1QTNF1–AS1 LNA1) = 69; n ( C1QTNF1–AS1 LNA2) = 72; n ( C1QTNF1–AS1 LNA3) =93). ( D ) Representative images of metaphase HCT116 cells stained for kinetochores (CREST, cyan), SAC (Mad2, green), and DNA (Hoescht, magenta) following siRNA-mediated depletion of RSRC2. ( E ) Insets show examples of MAD2 and CREST staining from RSRC2-depleted cells, as in panel (D); scale bar: 0.5 µm. ( F ) Characterization of SAC activity in HCT116 cells after siRNA-mediated depletion of RSRC2. Percentage of mitotic cells with Mad2 signal on misaligned chromosomes using the same antibodies as in panel (C); N = 4 ( n (Ctl si) = 108; n (RSRC2 si) = 126). ( G ) Quantification of mitotic duration from time-lapse imaging microscopy following LNA-mediated depletion of C1QTNF1–AS1 (left panel) and siRNA-mediated depletion of RSRC2 (right panel) in HCT116 H2B–GFP cells treated with nocodazole. N = 3 ( n (Ctl A) = 49; n ( C1QTNF1–AS1 LNA1) = 47; n ( C1QTNF1–AS1 LNA2) = 50; n ( C1QTNF1–AS1 LNA3) =51, n (Ctl si) = 87; n (RSRC2 si) = 51). ( H ) Changes in mitotic index after LNA-mediated depletion of C1QTNF1–AS1 (left panel) and siRNA-mediated depletion of RSRC2 (right panel) in HCT116 cells. Percentage of cells in mitosis shown based on Histone–H3–pS10 staining; N = 3 ( n (Ctl A) = 119; n ( C1QTNF1–AS1 LNA1) = 141; n ( C1QTNF1–AS1 LNA3) = 136; n (Ctl si) = 129; n (RSRC2 si) = 204). Error bars in all panels are shown as mean ± S.E.M.; scale bar: 5 μm. An unpaired t -test with Welch’s correction was applied in panels (C) and (F). The Mann–Whitney test was used in panel (G). A one-way ANOVA (left panel) and an unpaired t -test (right panel) were used in panel (H).* <0.05, **<0.01, ***<0.001 and ****<0.0001.

Article Snippet: Human normal retinal pigment epithelial hTERT-RPE1 (ATCC) and hTERT-RPE1 H2B-GFP (provided by Prof. David Pellman, USA) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) F12 medium (Sigma, D8437), supplemented with 10% foetal bovine serum (FBS, A5256801, Gibco).

Techniques: Staining, Activity Assay, Imaging, Microscopy, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: G protein Gα q subunits engage targets in the nucleus involved in chromatin remodeling and gene expression

doi: 10.1016/j.jbc.2026.111322

Figure Lengend Snippet: Activation-dependent Gα q translocation into the nucleus. A , f-Gα q localizes strongly to the PM while ( B ) SMARCD3-V5 localizes strongly to the nucleus in HEK293 cells. C , constitutive activation of Gα q enhances nuclear residency and is prevented by tethering Gα q to the PM. HEK293 cells stably expressing Histone H2B-HiBiT were transfected with the indicated Gα q -LgBiT cDNA containing plasmids. In parallel cells were co-transfected with the indicated Gα q -LgBiT cDNAs and GFP-HiBiT to account for differences in expression. Twenty four hour later cells were assessed for luminescence complementation. Data are corrected for differences in expression based on GFP HiBiT. N = 7. Data were analyzed using a one-way ANOVA with Sidak’s multiple comparisons post-test (relevant p -values shown). Raw luminescence values: GFP-HiBiT: 20 × 10 6 ± 5 × 10 6 (SEM), N = 3 vs. H2B HiBiT: 0.22 ×10 6 ± 0.08 × 10 6 , N = 7, indicate 1% of the Gα q is present in the nucleus. D , activation of M3 muscarinic receptors enhances Gα q -LgBiT nuclear accessibility. HEK293 cells stably expressing Histone H2B-HiBiT were transfected with Gα q (wt) -LgBiT and M3 muscarinic receptor cDNA plasmids. Twenty four hour later cells were stimulated for 3 h with the indicated concentrations of carbachol followed by measurement of luminescence complementation.

Article Snippet: H2B-HiBiT was synthesized by Twist Biosciences and was designed with the HiBiT sequence (VSGWRLFKKIS) followed by the V5 Tag, followed by H2B and inserted into pTwistCMV.

Techniques: Activation Assay, Translocation Assay, Stable Transfection, Expressing, Transfection

Journal: Science Advances

Article Title: Insights into the structure and evolution of the human SAGA complex by affinity-ligand purification

doi: 10.1126/sciadv.aec8104

Figure Lengend Snippet: ( A ) Proteomic analysis of the purified SAGA and ATAC complexes. For each identified protein the table shows the normalized spectral abundance factor (NSAF) value calculated from the PSMs divided by the number of amino acids (AA), and the normalization of the NSAF value to SUPT20H as rough estimation of stoichiometry. SAGA subunits are colored purple, ATAC subunits are colored blue, purple bold represents the common subunits between SAGA and ATAC, and copurified bromodomain containing proteins are in green. ( B ) Western blot analysis of different purification steps detecting the TADA2B subunit of SAGA. ( C ) Relative quantification of the detected bands in (B). ( D ) Colloidal Coomassie blue–stained SDS-PAGE of the purified SAGA and ATAC complexes; the identity of indicated bands was verified by MS. ( E ) Deubiquitination activity of the purified SAGA on the fluorogenic ubiquitin–amino methyl coumarin (Ub-AMC) over time. ( F ) Acetyltransferase activity of the purified SAGA and ATAC complex on nucleosome core particles (NCPs). Acetylation of histone was detected by a pan-acetyl-lysine antibody. Anti-GCN5 and histone H2B antibodies were used as loading control of SAGA/ATAC and NCPs, respectively.

Article Snippet: The primary antibodies used are against TADA2B (produced in-house, no. 3122), GCN5 (produced in-house, no. 2676), and histone H2B (Cell Signaling, 2934S).

Techniques: Purification, Western Blot, Quantitative Proteomics, Staining, SDS Page, Activity Assay, Ubiquitin Proteomics, Control