rabbit anti c peptide  (Cell Signaling Technology Inc)

Journal: Nucleic Acids Research

Article Title: AURKB-driven dissolution of CIZ1–RNA assemblies from the inactive X chromosome in mitosis

doi: 10.1093/nar/gkag018

Figure Lengend Snippet: Dissolution of CIZ1–Xi assemblies in mitosis. ( A ) Model of CIZ1–RNA assemblies surrounding and protecting the modification status of underlying chromatin [ , ]. ( B ) Illustrative immunofluorescence images of female murine D3T3 cells stained for CIZ1 via N-terminal epitopes (CIZ1-N, red, detected with pAb 1794), revealing large protein assemblies at the inactive X chromosome (white arrows) that are not detected in mitosis. DNA is shown in blue, bar is 5 µm. ( C ) Diagram illustrating loss in mitosis and the early G1 phase window during which reformation of CIZ1–Xi assemblies takes place, determined previously using cells that were synchronized in G1 phase by release from arrest with nocodazole . ( D ) Map showing conserved putative AURKB phosphorylation sites between murine and human CIZ1 (circles) displayed on full-length murine CIZ1 ( NP_082688.1 .) The location of epitopes of CIZ1 antibodies used throughout is shown above. Conserved prion-like domains (PLD1 and PLD2) are in red , zinc fingers 1–3 in cyan (ZnF_C2H2 SM00355, ZF_C2H2 sd00020, and ZF_C2H2 sd00020), acidic region (Ac) in yellow, matrin-3 homology domain (MH3) in orange (ZnF_U1, smart00451), and h37/m38 amino-acid C-terminal tail in blue. The sequence context and identity of three conserved AURKB phosphorylation sites in the extreme C-terminus are shown. ( E ) Frequency of cells with CIZ1–Xi assemblies (red) or nucleus-wide SAFA (blue) in cells passing through the stages of mitosis indicated, for D3T3 cells and female primary embryonic fibroblasts (PEFs at p3), in the presence and absence of the AURKB kinase inhibitor barasertib at 0.1 and 1 µM. Results show the average of 3–4 independent replicates within one experiment for each line, with SEM. n indicates the number of nuclei inspected (PEF grey, 3T3 black). Statistical analysis of CIZ1–Xi frequency in anaphase cells shows one-way ANOVA with Tukey post hoc test within each cell type, where * <.05, ** <.01, *** <.001. Below, example immunofluorescence images of D3T3 cells through mitosis, with and without 1 µM barasertib. Cells were stained for the N-terminal domains of CIZ1 (CIZ1-N, red) and SAFA (green). DNA is shown in blue, bar is 5 microns. ( F ) Upper histogram shows the proportion of cells with CIZ1-marked Xi in cycling populations of female D3T3 cells after the indicated times exposed to 300 nM Okadaic acid, visualized via CIZ1-N (red). Lower, histograms show the effect of the indicated concentrations of tautomycin for 15 h, stained for CIZ1-N or the ‘tail’ epitope in the C-terminal end of CIZ1 (CIZ1-C, rabbit pAb). Comparison of technical replicates is by t-test, where * <.05, ** <.01, *** <.001. Error bars show SEM. Below, example images showing H3K27me3-marked Xi chromatin in cells stained for CIZ1-N or CIZ1-C at 15 h with or without tautomycin. Bar is 5 microns.

Article Snippet: CIZ1 (C-terminal tail) Rabbit anti-peptide , Novus (NB100-74624).

Techniques: Dissolution, Modification, Immunofluorescence, Staining, Phospho-proteomics, Zinc-Fingers, Sequencing, Comparison

Journal: Nucleic Acids Research

Article Title: AURKB-driven dissolution of CIZ1–RNA assemblies from the inactive X chromosome in mitosis

doi: 10.1093/nar/gkag018

Figure Lengend Snippet: The C-terminal tail specifies nuclear immobilization and limits aggregation. ( A ) Immunofluorescence images showing female murine fibroblasts transfected with full-length mouse GFP-845, or derived constructs m845 Δ15 (lacking the C-terminal 15 amino acids), m845 DDD or m845 AAA (green). Cells were co-stained for H3K27me3 (red). DNA is shown in blue, bar is 10 microns. ( B ) Frequency of nuclei containing large (non-Xi) CIZ1 aggregates, shown as percentage of cells transfected. Results are shown with (+) and without (−) prefixation detergent wash (Det.). n = transfected nuclei counted. ( C ) Schematic showing the C-terminal portion of CIZ1, GFP-mC275 and derived GFP-mC275 DDD , and their use in 48 h transient expression experiments to assess their ability to assemble into detergent-resistant structures . Below, example images of transfected nuclei. Histograms show retention frequency in male (N = 2) and female (N = 2) CIZ1 null primary embryonic fibroblasts that were transfected (green). n denotes technical replicates for each cell population, with number of nuclei scored in parentheses. ( D ) Illustration displaying the effect of AURKB site cluster phosphomimic (P) on CIZ1’s association with chromatin and associated detergent-resistant nuclear structures.

Article Snippet: CIZ1 (C-terminal tail) Rabbit anti-peptide , Novus (NB100-74624).

Techniques: Immunofluorescence, Transfection, Derivative Assay, Construct, Staining, Expressing

Journal: Nucleic Acids Research

Article Title: AURKB-driven dissolution of CIZ1–RNA assemblies from the inactive X chromosome in mitosis

doi: 10.1093/nar/gkag018

Figure Lengend Snippet: AURKB site modification in mitosis. ( A ) Western blot showing denatured proteins in whole cell lysates collected from untransfected D3T3 cells, or populations expressing full-length mouse GFP-m845 WT or GFP-m845 DDD , after immunostaining for CIZ1-N or CIZ1-C (tail epitope), or β-actin, and GFP as indicated. ( B ) Western blot showing denatured endogenous proteins in chemically treated D3T3 cells to achieve cell cycle enrichment in mitosis (M, nocodazole), S phase (S, thymidine), or a phosphatase-suppressed state (okadaic acid). Immunoblotting for C-terminal tail and N-terminal CIZ1 indicates a reduction in tail epitope, compared to untreated cells, during arrest in metaphase, or after phosphatase inhibition. Histone H3 is shown as a loading control. Cy, cycling. ( C ) Illustration showing data interpretation in which the CIZ1 tail AURKB site cluster is phosphorylated in mitosis, driving dispersal of CIZ1 from Xi assemblies. ( D ) Female D3T3 cells in stages of mitosis as indicated, immunostained for CIZ1-N or CIZ1-C and co-stained for SAFA. Right, histograms show frequency of retention in interphase (I), prophase (P), metaphase (M), or anaphase (A), where N indicates replicate analyses and n nuclei scored. Lower, by metaphase CIZ1-N and CIZ1-C are significantly different ( P < .00017), student’s t-test. ( E ) Experimental overview of in vitro kinase reactions using purified recombinant human CIZ1 C-terminal fragment C179 and purified AURKB. Middle, products analysed by western blot with C-terminal CIZ1 epitope-defined antibodies, showing changes in reactivity in response to exposure to increasing concentrations of AURKB kinase. Graph shows band intensities relative to untreated C179 control. Products were also analysed by mass spectrometry .

Article Snippet: CIZ1 (C-terminal tail) Rabbit anti-peptide , Novus (NB100-74624).

Techniques: Modification, Western Blot, Expressing, Immunostaining, Inhibition, Control, Staining, In Vitro, Purification, Recombinant, Mass Spectrometry

Journal: Nucleic Acids Research

Article Title: AURKB-driven dissolution of CIZ1–RNA assemblies from the inactive X chromosome in mitosis

doi: 10.1093/nar/gkag018

Figure Lengend Snippet: CIZ1 C-terminal interaction partners. ( A ) Overview of nuclear protein interaction studies using mammalian cell nuclear extracts from HeLa cells and recombinant GST-tagged hCIZ1 C-terminal fragment C179. Domains coloured as in Fig. . Below, volcano plots showing protein interaction partners identified with high confidence in three independent studies and their relative retrieval by GST-hC179 compared to GST control. Significance (−log 10 FDR q -value) is plotted against log 2 fold change (FC), derived from n = 4 replicates in each case. Significant interaction partners are ≥2-fold more abundant in CIZ1–retrieved samples compared to GST samples, at q -value ≤0.05. Data are given in . ( B ) Venn diagram showing common interaction partners between three independent studies. The 56 core interaction partners are listed in . ( C ) CIZ1 interaction partners in common with 80 Xist interactors identified by CHIRP-MS or iDRIP . Venn diagram indicates those that interact with CIZ1 in all three of our studies. Proteins that were common to both Xist studies and also identified in any of our CIZ1 interaction studies are listed in . ( D ) Simplified STRING diagram showing 56 core CIZ1 interaction partners, clustered using MCL clustering. Three unclustered proteins (dark pink) and one chromatin protein (green) are nuclear matrix-associated proteins . ( E ) Individual abundance (mean of four replicates) across three studies for four nuclear matrix proteins in the core 56 interaction list (SAFB2, SMARCA5, NUMA1, SLTM) and two that appear in at least one of the studies (SAFA, SAFB1). Histograms show fraction of high-confidence peptides in bait and control for each study. ( F ) Example immunofluorescence images of SAFB2 (green) in cycling WT and CIZ1 null PEFs, co-stained for CIZ1-N (red), and DAPI (blue). Box and whisker plots show intensity measures derived from two independent primary cell populations (N) for each genotype. n = number of nuclei measured. Comparison is by t-test, where *** denotes P < .001 and indicates a significant reduction of bound SAFB2 epitope in CIZ1 null cells. ( G ) As in panel (F) but for SAFA, which is not significantly changed in CIZ1 null cells.

Article Snippet: CIZ1 (C-terminal tail) Rabbit anti-peptide , Novus (NB100-74624).

Techniques: Recombinant, Control, Derivative Assay, Immunofluorescence, Staining, Whisker Assay, Comparison

Journal: Nucleic Acids Research

Article Title: AURKB-driven dissolution of CIZ1–RNA assemblies from the inactive X chromosome in mitosis

doi: 10.1093/nar/gkag018

Figure Lengend Snippet: Effect of mutation on CIZ1 C-terminal interaction partners. ( A ) Volcano plots displaying protein interaction partners, comparing WT human C179 with C179 DDD or C179 Δtail in independent studies. The majority of proteins were unaffected. Those significantly increased or decreased are highlighted (log 2 fold change ±1, q ≤ 0.05). See also . To the right, STRING diagrams showing functional protein clusters for differentially retrieved proteins. Cluster identities are given in . ( B ) Summary table describing effects of mutations on study-specific interaction partners and core 56 interaction partners (in parentheses). ( C ) Heatmaps showing peptide abundance of the core 56 interaction partners retrieved in control (GST), WT (C179), and mutant reactions, as indicated ( n = 4 replicates). Upper, 10 proteins that are significantly reduced in C179 DDD compared to WT are indicated. Lower, of the core 56 only 5 are significantly changed [see panel (D)]. ( D ) Plot displays log 2 FC of core 56 proteins for C179 DDD (blue) and C179 Δtail (orange), with identities. Ten proteins that are significantly reduced upon phosphomimetic mutation are labelled in blue. Nuclear matrix proteins SAFB2, SLTM, and SMARCA5 are not affected; however, NUMA1 was increased in C179 Δtail (green).

Article Snippet: CIZ1 (C-terminal tail) Rabbit anti-peptide , Novus (NB100-74624).

Techniques: Mutagenesis, Functional Assay, Control

Journal: Nucleic Acids Research

Article Title: AURKB-driven dissolution of CIZ1–RNA assemblies from the inactive X chromosome in mitosis

doi: 10.1093/nar/gkag018

Figure Lengend Snippet: Interaction between CIZ1 dimer and RNA is regulated by AURKB sites in the C-terminal tail. ( A ) Schematic of h/m CIZ1 showing conserved domains, in yellow (acidic domain), orange (MH3 dimerization domain), and blue (unstructured tail h37/m38 C-terminal amino acids). Below, human C-terminal (C179) fragments, and derived mutants used as bait fragments in interaction studies, including C179 Δtail and phosphomimic C179 DDD . Below, C-terminal fragment encompassing the Zn finger motifs used for modelling (green, C305). Left, SDS–PAGE gels showing purified protein preparations stained with Coomassie Blue, or probed with anti-CIZ1 mAb 87, which recognizes all three proteins, or anti-CIZ1 tail pAb, which recognizes an epitope deleted in C179 Δtail and mutated in C179 DDD . ( B ) SEC-MALLS, showing normalized UV absorbance at 280 nm and molar mass (dotted line) for human CIZ1-C179 (blue), and human CIZ1-C179 Δtail (orange). ( C ) SEC-MALLS chromatogram showing normalized UV absorbance at 280 nm and molar mass (dotted line) for equivalent murine fragment C181 (black), and derived deletion mutant lacking the matrin 3 homology domain (C181 ΔMH3 , yellow). ( D ) Summary of measured molecular masses, indicating that the C-terminal fragment forms a stable dimer that is dependent on the MH3 domain but not the tail region. ( E ) AlphaFold dimer structure predictions of MH3 domain, showing human CIZ1 aa 779–838 uniprot Q9ULV3-1 (blue) and murine CIZ1 aa 725–785 uniprot Q8VEH2 (cyan). The domain forms a tight dimer with monomer–monomer interactions involving main chain hydrogen bonding between β-strands of the two MH3-type Zn finger motifs. ( F ) Example electrophoretic mobility shift assays (EMSA) showing the effect of C179, C179 Δtail , and C179 DDD on the mobility of digoxygenin (DIG)-labelled Xist repeat E RNA probe (left, 0.66 nM) or GAPDH RNA (right, 0.65 nM). Below, immunoblots of EMSA membranes using CIZ1 anti-MH3 domain antibody. Above, murine Xist structure and the derived Xist repeat E RNA probe used in EMSAs. Right, quantification of binding based on the fraction of shifted probe, derived from three replicate experiments (see also ). Graphs show means ± SEM. ( G ) AlphaFold-Multimer [ , ] structure prediction of human C-terminal aa 592–898 (hC306), showing the highest-ranking prediction, in which the acidic domains (yellow) are exposed and the unstructured tails (blue) extend from the core. ( H ) Model, depicting CIZ1 homodimers interacting with chromosome-associated RNAs via its C-terminal tails, with N-terminal PLD domains available for association with other proteins or other RNAs (left). Right, shows AURKB-mediated phosphorylation driving release from chromosome-associated RNA. In vitro in interphase this results in PLD-driven CIZ1 aggregation.

Article Snippet: CIZ1 (C-terminal tail) Rabbit anti-peptide , Novus (NB100-74624).

Techniques: Derivative Assay, SDS Page, Purification, Staining, Mutagenesis, Electrophoretic Mobility Shift Assay, Western Blot, Binding Assay, Phospho-proteomics, In Vitro