ring1b mouse mab  (Proteintech)

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: L3MBTL2 maintains MYCN-amplified neuroblastoma cell proliferation through silencing NRIP3 and BRME1 genes.

doi: 10.1111/gtc.13148

Figure Lengend Snippet: FIGURE 7 L3MBTL2-knockout reduces the level of H2AK119ub around the NRIP3 and BRME1 transcription start sites (TSSs). (a, b) Representative integrative genomics viewer (IGV) screenshots showing ChIP-seq-data-derived peaks of L3MBTL2, PCGF6, RING1B, H2AK119ub, H3K27me3, and MYCN on the NRIP1 and BRME1 loci. The blue boxes below each peak indicate peaks called using hiddenDomains with default parameters. The black boxes indicate the range used for ChIP-qPCR primer design. (c) Heatmap of PCGF6, MYCN, and H2AK119ub ChIP-seq signals in control (left) or L3MBTL2-knockout (right). PCGF6 ChIP-seq signal intensities in controls are sorted in descending order. (d) ChIP-qPCR showing changes in PCGF6, MYCN, and H2AK119ub binding around the NRIP3 and BRME1 promoter regions upon L3MBTL2 depletion. Numbers in parentheses indicate the primer sets used in Table S2. Data are presented as the mean ± SD, N = 3 (two-tailed Student's t-test).

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense precipitated using normal rabbit IgG (Wako 148-09551), L3MBTL2 rabbit pAb (Active Motif), RING1B mouse mAb (Atsuta et al., 2004), ubiquityl-histone H2A (Lys119) (CST D27C4) rabbit mAb, or PCGF6 rabbit pAb (Proteintech, 24103-1-AP) with Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Chicago, IL, USA).

Techniques: Knock-Out, ChIP-sequencing, Derivative Assay, ChIP-qPCR, Control, Binding Assay, Two Tailed Test

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) Schematic representation of CBX2-PRC1. (B) A hypothetical model describing how CBX2-PRC1 is assembled into condensates through phase separation. (C) Representative epi-fluorescence images of condensates of individual CBX2-PRC1 components. Scale bars, 5.0 μm. (D) Condensed fraction of CBX2 and PHC1/2/3 quantified from (C). Error bars denote SD. (E–G) Representative epi-fluorescence images of the scaffold CBX2 and the clients. CBX2, at a fixed concentration of 0.5 μM, was mixed with serial dilutions of the clients RING1B (E), MEL18 (F), and PHC1 (F). Scale bars, 5.0 μm. (H–M) Condensed fraction (H–J) and condensate size (K–M) of the scaffold CBX2 and the clients quantified from (E)–(G). Error bars denote SD.

Article Snippet: Anti-RING1B mouse mAb , MBL , Cat#D139-3; RRID: AB_592650.

Techniques: Fluorescence, Concentration Assay

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) Schematic representation of the CBX-PRC1 complexes. (B) Condensed fraction of the CBX proteins quantified from . Error bars denote SD. (C) Representative epi-fluorescence images of the CBX-PRC1 components. Panels on left: RING1B was unlabeled and not shown. Panels on right: only the RING1B images are shown. Scale bars, 5.0 μm. (D) Box plot of condensed fraction quantified from (C). (E) A hypothetical model describing how individual CBX-PRC1 complexes are assembled to condensates in vitro . (F) Live-cell epi-fluorescence images showing subnuclear localization of the CBX proteins fused with HaloTag treated with and without Dox. Scale bars, 5.0 μm. (G) Box plot of condensed fraction of the CBX proteins quantified from (F). p value is calculated using Student’s t test (**p < 0.01).

Article Snippet: Anti-RING1B mouse mAb , MBL , Cat#D139-3; RRID: AB_592650.

Techniques: Fluorescence, In Vitro

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) Schematic representation for CRISPR-Cas9-mediated homologous recombination to insert HaloTag to the C terminus of Cbx2 in mESCs. Scissors indicate the sgRNA-targeted location. Red arrows indicate the primers used to verify the insertion. LHA, left homology arm; RHA, right homology arm; HT, HaloTag. (B) Agarose gel analysis of PCR amplicons from homozygous HaloTag ( Cbx2 HT/HT ) and heterozygous HaloTag ( Cbx2 WT/HT ) knockin mESC lines. Arrows show the correct size of PCR amplicons. (C) Western blots for HaloTag comparing wild-type (WT), homozygous CBX2-HT, and heterozygous CBX2-HT mESC lines. (D) Live-cell imaging showing subnuclear distribution of CBX2-HT in Cbx2 HT/HT (left) and Cbx2 HT/WT (right) mESC lines. The number of condensates is shown to the right, along with the intensity ratio of condensates to non-condensed regions and the condensed fraction. Scale bars, 5.0 μm. (E) Co-immunostaining analysis of CBX2-HT as well as endogenous RING1B and PHC1 in Cbx2 HT/HT cell line. CBX2-HT was stained by an anti-HaloTag antibody. RING1B and PHC1 were stained by anti-RING1B and anti-PHC1 antibodies, respectively. Scale bars, 5.0 μm. (F) Live-cell imaging showing subnuclear localization of the CBX2-PRC1 components. The expression level is controlled by a tetracycline-response element (TETp, top panel) and induced by doxycycline (Dox). The thinness of the arrow corresponds to the level of expression. Scale bars, 5.0 μm. (G) Box plot of condensed fraction of the CBX2-PRC1 components quantified from (F). p value is calculated using Student’s t test (*p < 0.05; **p < 0.01).

Article Snippet: Anti-RING1B mouse mAb , MBL , Cat#D139-3; RRID: AB_592650.

Techniques: CRISPR, Homologous Recombination, Agarose Gel Electrophoresis, Knock-In, Western Blot, Live Cell Imaging, Immunostaining, Staining, Expressing

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) A hypothetical model describing how condensate composition regulates the partitioning of CBX2-PRC1 components and nucleosomes and the exchange properties of the scaffold CBX2. Colored hexagons are the CBX2-PRC1 clients (magenta) and nucleosomes (green). (B) Representative epi-fluorescence images of CBX2-PRC1 subunits in the four-component (CBX2, RING1B [R], MEL18 [M], and PHC1 [P]) system. Scale bars, 5.0 μm. (C) Box plot of condensed fraction in the four-component system quantified from (B). p value is calculated using Student’s t test (*p < 0.05; **p < 0.01). (D) FRAP curves of CBX2 in the single-component, two-component, three-component, and four-component systems. Error bars denote SD. (E) Example confocal fluorescence images of CBX2 and nucleosomes (Nuc.) in the two-component, three-component, four-component, and five-component systems. Scale bars, 5.0 μm. (F) Box plot of condensed fraction of CBX2 and nucleosomes quantified from (E). p value is calculated using Student’s t test (**p < 0.01). (G) FRAP curves of YFP-CBX2 in the two-, three-, four-, and five-component systems. Error bars denote SD. (H) Representative live-cell epi-fluorescence images of HT-CBX2 in wild-type (WT), Ring1a −/− /b −/− , and Bmi1 −/− /Mel18 −/− mESC lines. Scale bars, 5.0 μm. (I and J) Box plots of condensed fraction (I) and size (J) of HT-CBX2 condensates quantified from (H). p value is calculated using Student’s t test (**p < 0.01). Error bars denote SD. (K) Example confocal images of FRAP of HT-CBX2 in wild-type (WT), Ring1a −/− /b −/− , and Bmi1 −/− /Mel18 −/− mESC lines. Red arrows show condensates to be bleached. Scale bar, 5.0 μm. (L) FRAP curves of HT-CBX2 within and outside condensates in wild-type (WT), Ring1a −/− /b −/− , and Bmi1 −/− /Mel18 −/− mESC lines. Error bars denote SD.

Article Snippet: Anti-RING1B mouse mAb , MBL , Cat#D139-3; RRID: AB_592650.

Techniques: Fluorescence

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: (A) Residue-resolution coarse-grained model of CBX2 and RING1B. f, folded regions; d, disordered regions. (B) Minimal coarse-grained model of CBX2, RING1B, MEL18, and PHC1 that describes the proteins as patchy colloids. CBX2 and PHC1 are represented as 4-valency patchy particles, while RING1B and MEL18 are represented as 3-valency patchy particles. The interaction matrix shows the relative pairwise interaction strengths between the patches on the four different proteins in the minimal model at reduced temperature T = 1 . (C) Contact maps showing the frequency of contacts between the different regions of the proteins, for a pure CBX2 system and a 1:1 CBX2/RING1B mixture. For the regions with a larger relative contribution of contacts highlighted by the red square, a residue-resolution contact map is shown. (D) Variation of T c of pure CBX2 and RING1B systems, as well as their mixtures with varying RING1B mole fraction using our residue-resolution model (purple) and minimal model (red). For each set of data points, the T c of each system relative to the T c of the pure CBX2 system is plotted. (E) Diffusion coefficients of CBX2 measured in direct-coexistence simulations of the dense phase of mixtures with different compositions at T = 1 .

Article Snippet: Anti-RING1B mouse mAb , MBL , Cat#D139-3; RRID: AB_592650.

Techniques: Residue, Diffusion-based Assay

Journal: Cell reports

Article Title: Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

doi: 10.1016/j.celrep.2023.113136

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-RING1B mouse mAb , MBL , Cat#D139-3; RRID: AB_592650.

Techniques: Virus, Recombinant, Staining, Modification, Saline, Live Cell Imaging, Electroporation, Purification, Protease Inhibitor, Membrane, Stripping Membranes, Bradford Assay, Plasmid Preparation, Software, Microscopy