ubiquityl histone h2b lys120 d11 rabbit mab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ubiquityl histone h2b lys120 d11 rabbit mab
    a Model structure of the RINGA-RINGB-Rad6A-ubiquitin complex bound to the nucleosome in two views. b Close-up view of ubiquitin and <t>H2B.</t> Two lysine residues (H2BK120 and H2BK116) near G76 of ubiquitin are shown. H2BS112, whose GlcNAcylation stimulates H2BK120 ubiquitination, is also shown. c Proposed mechanistic model. The wild-type Bre1 complex can bind to the nucleosome in two orientations, but H2BK120 ubiquitination occurs only when Bre1A binds to the acidic patch, as RING A , but not RING B , can recruit Rad6A and ubiquitin. Bre1B with G974T B -A978T B double substitution can recruit Rad6A and ubiquitin; thus, H2BK120 ubiquitination occurs in both binding modes.
    Ubiquityl Histone H2b Lys120 D11 Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Structure of the human Bre1 complex bound to the nucleosome"

    Article Title: Structure of the human Bre1 complex bound to the nucleosome

    Journal: Nature Communications

    doi: 10.1038/s41467-024-46910-8

    a Model structure of the RINGA-RINGB-Rad6A-ubiquitin complex bound to the nucleosome in two views. b Close-up view of ubiquitin and H2B. Two lysine residues (H2BK120 and H2BK116) near G76 of ubiquitin are shown. H2BS112, whose GlcNAcylation stimulates H2BK120 ubiquitination, is also shown. c Proposed mechanistic model. The wild-type Bre1 complex can bind to the nucleosome in two orientations, but H2BK120 ubiquitination occurs only when Bre1A binds to the acidic patch, as RING A , but not RING B , can recruit Rad6A and ubiquitin. Bre1B with G974T B -A978T B double substitution can recruit Rad6A and ubiquitin; thus, H2BK120 ubiquitination occurs in both binding modes.
    Figure Legend Snippet: a Model structure of the RINGA-RINGB-Rad6A-ubiquitin complex bound to the nucleosome in two views. b Close-up view of ubiquitin and H2B. Two lysine residues (H2BK120 and H2BK116) near G76 of ubiquitin are shown. H2BS112, whose GlcNAcylation stimulates H2BK120 ubiquitination, is also shown. c Proposed mechanistic model. The wild-type Bre1 complex can bind to the nucleosome in two orientations, but H2BK120 ubiquitination occurs only when Bre1A binds to the acidic patch, as RING A , but not RING B , can recruit Rad6A and ubiquitin. Bre1B with G974T B -A978T B double substitution can recruit Rad6A and ubiquitin; thus, H2BK120 ubiquitination occurs in both binding modes.

    Techniques Used: Binding Assay

    anti ubiquityl histone h2b lys120  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti ubiquityl histone h2b lys120
    Anti Ubiquityl Histone H2b Lys120, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti ubiquityl histone h2b lys120 d11 xp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti ubiquityl histone h2b lys120 d11 xp
    Anti Ubiquityl Histone H2b Lys120 D11 Xp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti ubiquityl histone h2b lys120 d11 xp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti ubiquityl histone h2b lys120 d11 xp
    Low levels of H2BK123ub1 are detected in the absence of Lge1. ( A , B ) Immunoblots for histone H2BK123ub1 and/or histone H3K4 methylation (mono, me1; di, me2; tri, me3) in extracts prepared from ( A ) strains lacking Rad6, Bre1 or Lge1 and ( B ) strains lacking Rad6, Bre1 or Lge1 in the background of ubp8Δubp10Δ double deletion mutant. Ponceau S staining and histone H3 levels served as loading controls. Histone H2BK123ub1 was detected using <t>anti-H2B</t> or anti-H2BK120 ubiquityl antibody. Molecular weights of the protein standards used as size markers (kDa) are indicated.
    Anti Ubiquityl Histone H2b Lys120 D11 Xp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Structure–function analysis of histone H2B and PCNA ubiquitination dynamics using deubiquitinase-deficient strains"

    Article Title: Structure–function analysis of histone H2B and PCNA ubiquitination dynamics using deubiquitinase-deficient strains

    Journal: Scientific Reports

    doi: 10.1038/s41598-023-43969-z

    Low levels of H2BK123ub1 are detected in the absence of Lge1. ( A , B ) Immunoblots for histone H2BK123ub1 and/or histone H3K4 methylation (mono, me1; di, me2; tri, me3) in extracts prepared from ( A ) strains lacking Rad6, Bre1 or Lge1 and ( B ) strains lacking Rad6, Bre1 or Lge1 in the background of ubp8Δubp10Δ double deletion mutant. Ponceau S staining and histone H3 levels served as loading controls. Histone H2BK123ub1 was detected using anti-H2B or anti-H2BK120 ubiquityl antibody. Molecular weights of the protein standards used as size markers (kDa) are indicated.
    Figure Legend Snippet: Low levels of H2BK123ub1 are detected in the absence of Lge1. ( A , B ) Immunoblots for histone H2BK123ub1 and/or histone H3K4 methylation (mono, me1; di, me2; tri, me3) in extracts prepared from ( A ) strains lacking Rad6, Bre1 or Lge1 and ( B ) strains lacking Rad6, Bre1 or Lge1 in the background of ubp8Δubp10Δ double deletion mutant. Ponceau S staining and histone H3 levels served as loading controls. Histone H2BK123ub1 was detected using anti-H2B or anti-H2BK120 ubiquityl antibody. Molecular weights of the protein standards used as size markers (kDa) are indicated.

    Techniques Used: Western Blot, Methylation, Mutagenesis, Staining

    Substitution of alanine 120 in the H2B C-terminal helix with charged amino acid interferes with ubiquitination at lysine 123. ( A ) Sequences of the distal end of WT H2B C-terminal helix and of mutants. Lysine 123, the site of monoubiquitination is indicated. ( B ) Growth assay conducted by spotting tenfold serial dilutions of indicated strains on synthetic medium lacking histidine (− HIS) or lacking histidine and containing 5-fluoroorotic acid (− HIS + FOA). ( C , D ) Left: Immunoblots for H2BK123ub1 in extracts prepared from ( C ) UBP8UBP10 or ( D ) ubp8Δubp10Δ strains expressing Flag epitope-tagged WT or mutant histone H2B. Triangles denote increasing amounts of extracts used. Ponceau S staining served as loading control. Right: Fold-change in H2BK123ub1 levels in the indicated mutants relative to WT H2B (set as 1). For densitometry quantitation, the signals for H2BK123ub1 in WT or mutant H2B were initially normalized to the signals for Ponceau S-stained proteins. Plotted are means ± SEM from three independent experiments. ns not significant; *p -value < 0.05 (Student’s t-test).
    Figure Legend Snippet: Substitution of alanine 120 in the H2B C-terminal helix with charged amino acid interferes with ubiquitination at lysine 123. ( A ) Sequences of the distal end of WT H2B C-terminal helix and of mutants. Lysine 123, the site of monoubiquitination is indicated. ( B ) Growth assay conducted by spotting tenfold serial dilutions of indicated strains on synthetic medium lacking histidine (− HIS) or lacking histidine and containing 5-fluoroorotic acid (− HIS + FOA). ( C , D ) Left: Immunoblots for H2BK123ub1 in extracts prepared from ( C ) UBP8UBP10 or ( D ) ubp8Δubp10Δ strains expressing Flag epitope-tagged WT or mutant histone H2B. Triangles denote increasing amounts of extracts used. Ponceau S staining served as loading control. Right: Fold-change in H2BK123ub1 levels in the indicated mutants relative to WT H2B (set as 1). For densitometry quantitation, the signals for H2BK123ub1 in WT or mutant H2B were initially normalized to the signals for Ponceau S-stained proteins. Plotted are means ± SEM from three independent experiments. ns not significant; *p -value < 0.05 (Student’s t-test).

    Techniques Used: Growth Assay, Western Blot, Expressing, FLAG-tag, Mutagenesis, Staining, Quantitation Assay

    An in vivo DUB deletion mutant-based approach for evaluating ubiquitin conjugation and deubiquitination. ( A ) The steady-state levels of ubiquitination of a protein in vivo are maintained by the actions of ‘writer’ E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases and ‘eraser’ DUBs. ( B ) In yeast S. cerevisiae , the in vivo steady-state levels of H2BK123ub1 in a nucleosome are maintained by the ‘writer’ complex comprised of Rad6 (E2), Bre1 (E3) and accessory/adapter protein Lge1, and two ‘eraser’ DUBs, Ubp10 and the SAGA complex-associated Ubp8. IDR, intrinsically disordered region; cc, coiled-coil domain. ( C ) In the ubp8Δubp10Δ double null mutant strain, H2BK123ub1 accumulates due to ubiquitin addition and absence of deubiquitination. ( D ) High levels of H2BK123ub1 are not observed in the ubp8Δubp10Δ mutant strain when either the IDR or coiled-coil domain of Lge1 or the C-terminal acidic tail of Rad6 are deleted. Thus, the absence of relevant DUBs revealed the roles for various regions or domains of proteins involved in the ubiquitin-conjugation step. ( E ) Residues of the H2B C-terminal helix (Cα) impact the activity of the E2-E3 complex and the DUBs by influencing their access to substrate K123 or its ubiquitin conjugated form, respectively. Aspartate substitution at residue 120 in H2B Cα inhibits the Rad6-Bre1-Lge1-mediated monoubiquitination of H2BK123 in both WT and ubp8Δubp10Δ strains. In contrast, arginine substitution at position 120 in H2B Cα promotes removal of the conjugated ubiquitin by Ubp8 and Ubp10, as evidenced by the reduced H2BK123ub1 in the H2BA120R mutation in a strain expressing these two DUBs and not in their absence. Thus, the use of the DUB deletion strain informed on the dynamics of deubiquitination in addition to the ubiquitin conjugation.
    Figure Legend Snippet: An in vivo DUB deletion mutant-based approach for evaluating ubiquitin conjugation and deubiquitination. ( A ) The steady-state levels of ubiquitination of a protein in vivo are maintained by the actions of ‘writer’ E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases and ‘eraser’ DUBs. ( B ) In yeast S. cerevisiae , the in vivo steady-state levels of H2BK123ub1 in a nucleosome are maintained by the ‘writer’ complex comprised of Rad6 (E2), Bre1 (E3) and accessory/adapter protein Lge1, and two ‘eraser’ DUBs, Ubp10 and the SAGA complex-associated Ubp8. IDR, intrinsically disordered region; cc, coiled-coil domain. ( C ) In the ubp8Δubp10Δ double null mutant strain, H2BK123ub1 accumulates due to ubiquitin addition and absence of deubiquitination. ( D ) High levels of H2BK123ub1 are not observed in the ubp8Δubp10Δ mutant strain when either the IDR or coiled-coil domain of Lge1 or the C-terminal acidic tail of Rad6 are deleted. Thus, the absence of relevant DUBs revealed the roles for various regions or domains of proteins involved in the ubiquitin-conjugation step. ( E ) Residues of the H2B C-terminal helix (Cα) impact the activity of the E2-E3 complex and the DUBs by influencing their access to substrate K123 or its ubiquitin conjugated form, respectively. Aspartate substitution at residue 120 in H2B Cα inhibits the Rad6-Bre1-Lge1-mediated monoubiquitination of H2BK123 in both WT and ubp8Δubp10Δ strains. In contrast, arginine substitution at position 120 in H2B Cα promotes removal of the conjugated ubiquitin by Ubp8 and Ubp10, as evidenced by the reduced H2BK123ub1 in the H2BA120R mutation in a strain expressing these two DUBs and not in their absence. Thus, the use of the DUB deletion strain informed on the dynamics of deubiquitination in addition to the ubiquitin conjugation.

    Techniques Used: In Vivo, Mutagenesis, Conjugation Assay, Activity Assay, Residue, Expressing

    anti ubiquityl histone h2b lys120 d11 xp  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti ubiquityl histone h2b lys120 d11 xp
    (A-B) Immunoblots for histone H2BK123ub1 and/or histone H3K4 methylation (mono, me1; di, me2; tri, me3) in extracts prepared from A) strains lacking Rad6, Bre1 or Lge1 and B) strains lacking Rad6, Bre1 or Lge1 in the background of ubp8Δubp10Δ double deletion mutant. Ponceau S staining and histone H3 levels served as loading controls. Histone H2BK123ub1 was detected using <t>anti-H2B</t> or anti-H2BK120 ubiquityl antibody. Molecular weights of the protein standards used as size markers (kDa) are indicated.
    Anti Ubiquityl Histone H2b Lys120 D11 Xp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A system for in vivo evaluation of protein ubiquitination dynamics using deubiquitinase-deficient strains"

    Article Title: A system for in vivo evaluation of protein ubiquitination dynamics using deubiquitinase-deficient strains

    Journal: bioRxiv

    doi: 10.1101/2023.06.18.545485

    (A-B) Immunoblots for histone H2BK123ub1 and/or histone H3K4 methylation (mono, me1; di, me2; tri, me3) in extracts prepared from A) strains lacking Rad6, Bre1 or Lge1 and B) strains lacking Rad6, Bre1 or Lge1 in the background of ubp8Δubp10Δ double deletion mutant. Ponceau S staining and histone H3 levels served as loading controls. Histone H2BK123ub1 was detected using anti-H2B or anti-H2BK120 ubiquityl antibody. Molecular weights of the protein standards used as size markers (kDa) are indicated.
    Figure Legend Snippet: (A-B) Immunoblots for histone H2BK123ub1 and/or histone H3K4 methylation (mono, me1; di, me2; tri, me3) in extracts prepared from A) strains lacking Rad6, Bre1 or Lge1 and B) strains lacking Rad6, Bre1 or Lge1 in the background of ubp8Δubp10Δ double deletion mutant. Ponceau S staining and histone H3 levels served as loading controls. Histone H2BK123ub1 was detected using anti-H2B or anti-H2BK120 ubiquityl antibody. Molecular weights of the protein standards used as size markers (kDa) are indicated.

    Techniques Used: Western Blot, Methylation, Mutagenesis, Staining

    (A) Sequences of the distal end of WT H2B C-terminal helix and of mutants. Lysine 123, the site of monoubiquitination is indicated. (B) Growth assay conducted by spotting 10-fold serial dilutions of indicated strains on synthetic medium lacking histidine (-HIS) or lacking histidine and containing 5-fluoroorotic acid (-HIS+FOA). (C-D) Left: Immunoblots for H2BK123ub1 in extracts prepared from C) UBP8UBP10 or D) ubp8Δubp10Δ strains expressing Flag epitope-tagged WT or mutant histone H2B. Ponceau S staining served as loading control. Right: Fold-change in H2BK123ub1 levels in the indicated mutants relative to WT H2B (set as 1). For densitometry quantitation, the signals for H2BK123ub1 in WT or mutant H2B were initially normalized to the signals for Ponceau S-stained proteins. Plotted are means ± SEM from three independent experiments. ns, not significant; *, p -value <0.05 (Student’s t-test).
    Figure Legend Snippet: (A) Sequences of the distal end of WT H2B C-terminal helix and of mutants. Lysine 123, the site of monoubiquitination is indicated. (B) Growth assay conducted by spotting 10-fold serial dilutions of indicated strains on synthetic medium lacking histidine (-HIS) or lacking histidine and containing 5-fluoroorotic acid (-HIS+FOA). (C-D) Left: Immunoblots for H2BK123ub1 in extracts prepared from C) UBP8UBP10 or D) ubp8Δubp10Δ strains expressing Flag epitope-tagged WT or mutant histone H2B. Ponceau S staining served as loading control. Right: Fold-change in H2BK123ub1 levels in the indicated mutants relative to WT H2B (set as 1). For densitometry quantitation, the signals for H2BK123ub1 in WT or mutant H2B were initially normalized to the signals for Ponceau S-stained proteins. Plotted are means ± SEM from three independent experiments. ns, not significant; *, p -value <0.05 (Student’s t-test).

    Techniques Used: Growth Assay, Western Blot, Expressing, FLAG-tag, Mutagenesis, Staining, Quantitation Assay

    (A) The steady-state levels of ubiquitination of a protein in vivo are maintained by the actions of ‘writer’ E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases and ‘eraser’ DUBs. (B) In yeast S. cerevisiae , the in vivo steady-state levels of H2BK123ub1 in a nucleosome are maintained by the ‘writer’ complex comprised of Rad6 (E2), Bre1 (E3) and accessory/adapter protein Lge1, and two ‘eraser’ DUBs, Ubp10 and the SAGA complex-associated Ubp8. IDR, intrinsically disordered region; cc, coiled-coil domain. (C) In the ubp8Δubp10Δ double null mutant strain, H2BK123ub1 accumulates due to ubiquitin addition and absence of deubiquitination. (D) High levels of H2BK123ub1 are not observed in the ubp8Δubp10Δ mutant strain when either the IDR or coiled-coil domain of Lge1 or the C-terminal acidic tail of Rad6 are deleted. Thus, the absence of relevant DUBs revealed the roles for various regions or domains of proteins involved in the ubiquitin-conjugation step. (E) Residues of the H2B C-terminal helix (Cα) impact the activity of the E2-E3 complex and the DUBs by influencing their access to substrate K123 or its ubiquitin conjugated form, respectively. Aspartate substitution at residue 120 in H2B Cα inhibits the Rad6-Bre1-Lge1-mediated monoubiquitination of H2BK123 in both WT and ubp8Δubp10Δ strains. In contrast, arginine substitution at position 120 in H2B Cα promotes removal of the conjugated ubiquitin by Ubp8 and Ubp10, as evidenced by the reduced H2BK123ub1 in the H2BA120R mutation in a strain expressing these two DUBs and not in their absence. Thus, the use of the DUB deletion strain informed on the dynamics of deubiquitination in addition to the ubiquitin conjugation.
    Figure Legend Snippet: (A) The steady-state levels of ubiquitination of a protein in vivo are maintained by the actions of ‘writer’ E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases and ‘eraser’ DUBs. (B) In yeast S. cerevisiae , the in vivo steady-state levels of H2BK123ub1 in a nucleosome are maintained by the ‘writer’ complex comprised of Rad6 (E2), Bre1 (E3) and accessory/adapter protein Lge1, and two ‘eraser’ DUBs, Ubp10 and the SAGA complex-associated Ubp8. IDR, intrinsically disordered region; cc, coiled-coil domain. (C) In the ubp8Δubp10Δ double null mutant strain, H2BK123ub1 accumulates due to ubiquitin addition and absence of deubiquitination. (D) High levels of H2BK123ub1 are not observed in the ubp8Δubp10Δ mutant strain when either the IDR or coiled-coil domain of Lge1 or the C-terminal acidic tail of Rad6 are deleted. Thus, the absence of relevant DUBs revealed the roles for various regions or domains of proteins involved in the ubiquitin-conjugation step. (E) Residues of the H2B C-terminal helix (Cα) impact the activity of the E2-E3 complex and the DUBs by influencing their access to substrate K123 or its ubiquitin conjugated form, respectively. Aspartate substitution at residue 120 in H2B Cα inhibits the Rad6-Bre1-Lge1-mediated monoubiquitination of H2BK123 in both WT and ubp8Δubp10Δ strains. In contrast, arginine substitution at position 120 in H2B Cα promotes removal of the conjugated ubiquitin by Ubp8 and Ubp10, as evidenced by the reduced H2BK123ub1 in the H2BA120R mutation in a strain expressing these two DUBs and not in their absence. Thus, the use of the DUB deletion strain informed on the dynamics of deubiquitination in addition to the ubiquitin conjugation.

    Techniques Used: In Vivo, Mutagenesis, Conjugation Assay, Activity Assay, Expressing

    anti h2bk120ub  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti h2bk120ub
    <t>H2BK120ub</t> is deficient in the Sertoli cells in the Amh-Rnf20 −/− mice. a Mass spectrometry detection of the ubiquitination of the H2B at K120 with the peptide HAVSEGTK(120)AVTK in the Rnf20 Flox/Flox . The MQ software was used to analyze the data from mass spectrometry. X axis, m/z; Y axis, the intensity of ions; y, the C-terminal fragment ion (Y series). b Ubiquitinated peptide information at the position of the K120 in the Rnf20 Flox/Flox . The ubiquitination modification site was not detected in the Amh-Rnf20 −/− . c Immunofluorescent analysis of SOX9, H2BK120ub, and DMRT1 on serial paraffin-sections in the Rnf20 Flox/Flox and the Amh-Rnf20 −/− testes at 7 days after birth and adult mice. The nuclei were stained with DAPI. TRITC signals represent the localization of H2BK120ub, while FITC signals showed the localization of SOX9 or DMRT1. The white squares in the panels correspond to the enlarged panels. Sn, Sertoli cells; Sg, spermatogonia. Scale bar, 10 μm
    Anti H2bk120ub, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "RNF20 is required for male fertility through regulation of H2B ubiquitination in the Sertoli cells"

    Article Title: RNF20 is required for male fertility through regulation of H2B ubiquitination in the Sertoli cells

    Journal: Cell & Bioscience

    doi: 10.1186/s13578-023-01018-2

    H2BK120ub is deficient in the Sertoli cells in the Amh-Rnf20 −/− mice. a Mass spectrometry detection of the ubiquitination of the H2B at K120 with the peptide HAVSEGTK(120)AVTK in the Rnf20 Flox/Flox . The MQ software was used to analyze the data from mass spectrometry. X axis, m/z; Y axis, the intensity of ions; y, the C-terminal fragment ion (Y series). b Ubiquitinated peptide information at the position of the K120 in the Rnf20 Flox/Flox . The ubiquitination modification site was not detected in the Amh-Rnf20 −/− . c Immunofluorescent analysis of SOX9, H2BK120ub, and DMRT1 on serial paraffin-sections in the Rnf20 Flox/Flox and the Amh-Rnf20 −/− testes at 7 days after birth and adult mice. The nuclei were stained with DAPI. TRITC signals represent the localization of H2BK120ub, while FITC signals showed the localization of SOX9 or DMRT1. The white squares in the panels correspond to the enlarged panels. Sn, Sertoli cells; Sg, spermatogonia. Scale bar, 10 μm
    Figure Legend Snippet: H2BK120ub is deficient in the Sertoli cells in the Amh-Rnf20 −/− mice. a Mass spectrometry detection of the ubiquitination of the H2B at K120 with the peptide HAVSEGTK(120)AVTK in the Rnf20 Flox/Flox . The MQ software was used to analyze the data from mass spectrometry. X axis, m/z; Y axis, the intensity of ions; y, the C-terminal fragment ion (Y series). b Ubiquitinated peptide information at the position of the K120 in the Rnf20 Flox/Flox . The ubiquitination modification site was not detected in the Amh-Rnf20 −/− . c Immunofluorescent analysis of SOX9, H2BK120ub, and DMRT1 on serial paraffin-sections in the Rnf20 Flox/Flox and the Amh-Rnf20 −/− testes at 7 days after birth and adult mice. The nuclei were stained with DAPI. TRITC signals represent the localization of H2BK120ub, while FITC signals showed the localization of SOX9 or DMRT1. The white squares in the panels correspond to the enlarged panels. Sn, Sertoli cells; Sg, spermatogonia. Scale bar, 10 μm

    Techniques Used: Mass Spectrometry, Software, Modification, Staining

    RNF20 deficiency in Sertoli cells impairs the Cldn11 transcription. a Scatterplots of differentially expressed genes. Red scatter, genes with significant up-regulated; blue scatter, genes with significant down-regulated; gray scatter, genes with no significant difference. X axis, Lg (WT FPKM) in the Rnf20 Flox/Flox ; Y axis, Lg (KO FPKM) in the Amh-Rnf20 −/− . b Gene ontology (GO) terms analysis of down-regulated genes in the Sertoli cells of the Amh-Rnf20 −/− testes. c, d Heatmaps showing the expression levels of down-regulated genes in the terms spermatogenesis ( c ) and cell adhesion ( d ) in the Sertoli cells of the Rnf20 Flox/Flox and the Amh-Rnf20 −/− . Color bar, Log 2 (FPKM). e Quantitative real-time PCR analysis of the genes Rnf20 and Cldn11 . The expression levels of the genes were related to Hprt expression. Relative levels, 2 −ΔCt ; T-tests were performed. *, p < 0.05, **, p < 0.01. f Western blot analysis of the expression levels of RNF20, CLDN11, and H2BK120ub proteins in adult mice. β-ACTIN was used as an internal control. g ChIP-PCR assays. The antibody specific for H2BK120ub was used in the ChIP analysis and primers were designed in the regions of promoter and exons of Cldn11 in the testes of the Rnf20 Flox/Flox and the Amh-Rnf20 −/− . The black graphs indicated the enriched levels in the Rnf20 Flox/Flox mice, while the white graphs indicated the levels in the Amh-Rnf20 −/− mice
    Figure Legend Snippet: RNF20 deficiency in Sertoli cells impairs the Cldn11 transcription. a Scatterplots of differentially expressed genes. Red scatter, genes with significant up-regulated; blue scatter, genes with significant down-regulated; gray scatter, genes with no significant difference. X axis, Lg (WT FPKM) in the Rnf20 Flox/Flox ; Y axis, Lg (KO FPKM) in the Amh-Rnf20 −/− . b Gene ontology (GO) terms analysis of down-regulated genes in the Sertoli cells of the Amh-Rnf20 −/− testes. c, d Heatmaps showing the expression levels of down-regulated genes in the terms spermatogenesis ( c ) and cell adhesion ( d ) in the Sertoli cells of the Rnf20 Flox/Flox and the Amh-Rnf20 −/− . Color bar, Log 2 (FPKM). e Quantitative real-time PCR analysis of the genes Rnf20 and Cldn11 . The expression levels of the genes were related to Hprt expression. Relative levels, 2 −ΔCt ; T-tests were performed. *, p < 0.05, **, p < 0.01. f Western blot analysis of the expression levels of RNF20, CLDN11, and H2BK120ub proteins in adult mice. β-ACTIN was used as an internal control. g ChIP-PCR assays. The antibody specific for H2BK120ub was used in the ChIP analysis and primers were designed in the regions of promoter and exons of Cldn11 in the testes of the Rnf20 Flox/Flox and the Amh-Rnf20 −/− . The black graphs indicated the enriched levels in the Rnf20 Flox/Flox mice, while the white graphs indicated the levels in the Amh-Rnf20 −/− mice

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    anti ubiquity histone h2b lys120  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti ubiquity histone h2b lys120
    Anti Ubiquity Histone H2b Lys120, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cat no 5546 rrid ab 10693452  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cat no 5546 rrid ab 10693452

    Cat No 5546 Rrid Ab 10693452, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Recycling of modified H2A-H2B provides short-term memory of chromatin states"

    Article Title: Recycling of modified H2A-H2B provides short-term memory of chromatin states

    Journal: Cell

    doi: 10.1016/j.cell.2023.01.007


    Figure Legend Snippet:

    Techniques Used: Recombinant, Purification, Western Blot, Software

    anti h2bub1 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti h2bub1 antibody
    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) <t>H2Bub1</t> and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).
    Anti H2bub1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis"

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2023.02.014

    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).
    Figure Legend Snippet: The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).

    Techniques Used: RNA Sequencing Assay, Infection, Expressing

    BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, Infection, Staining, Immunohistochemistry

    H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, ChIP-sequencing, Infection, Binding Assay

    TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, Western Blot, Immunofluorescence, Staining

    Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.
    Figure Legend Snippet: Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.

    Techniques Used: Injection, Western Blot, Expressing, Fluorescence, Immunofluorescence, Staining, Micro-CT

    Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis
    Figure Legend Snippet: Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis

    Techniques Used:

    anti h2bub1 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti h2bub1 antibody
    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) <t>H2Bub1</t> and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).
    Anti H2bub1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti h2bub1 antibody/product/Cell Signaling Technology Inc
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    anti h2bub1 antibody - by Bioz Stars, 2024-07
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    1) Product Images from "BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis"

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2023.02.014

    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).
    Figure Legend Snippet: The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).

    Techniques Used: RNA Sequencing Assay, Infection, Expressing

    BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, Infection, Staining, Immunohistochemistry

    H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, ChIP-sequencing, Infection, Binding Assay

    TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.
    Figure Legend Snippet: TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Techniques Used: Expressing, Western Blot, Immunofluorescence, Staining

    Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.
    Figure Legend Snippet: Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.

    Techniques Used: Injection, Western Blot, Expressing, Fluorescence, Immunofluorescence, Staining, Micro-CT

    Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis
    Figure Legend Snippet: Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis

    Techniques Used:

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    Cell Signaling Technology Inc ubiquityl histone h2b lys120 d11 rabbit mab
    a Model structure of the RINGA-RINGB-Rad6A-ubiquitin complex bound to the nucleosome in two views. b Close-up view of ubiquitin and <t>H2B.</t> Two lysine residues (H2BK120 and H2BK116) near G76 of ubiquitin are shown. H2BS112, whose GlcNAcylation stimulates H2BK120 ubiquitination, is also shown. c Proposed mechanistic model. The wild-type Bre1 complex can bind to the nucleosome in two orientations, but H2BK120 ubiquitination occurs only when Bre1A binds to the acidic patch, as RING A , but not RING B , can recruit Rad6A and ubiquitin. Bre1B with G974T B -A978T B double substitution can recruit Rad6A and ubiquitin; thus, H2BK120 ubiquitination occurs in both binding modes.
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    Cell Signaling Technology Inc anti ubiquityl histone h2b lys120
    a Model structure of the RINGA-RINGB-Rad6A-ubiquitin complex bound to the nucleosome in two views. b Close-up view of ubiquitin and <t>H2B.</t> Two lysine residues (H2BK120 and H2BK116) near G76 of ubiquitin are shown. H2BS112, whose GlcNAcylation stimulates H2BK120 ubiquitination, is also shown. c Proposed mechanistic model. The wild-type Bre1 complex can bind to the nucleosome in two orientations, but H2BK120 ubiquitination occurs only when Bre1A binds to the acidic patch, as RING A , but not RING B , can recruit Rad6A and ubiquitin. Bre1B with G974T B -A978T B double substitution can recruit Rad6A and ubiquitin; thus, H2BK120 ubiquitination occurs in both binding modes.
    Anti Ubiquityl Histone H2b Lys120, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti ubiquityl histone h2b lys120 d11 xp
    a Model structure of the RINGA-RINGB-Rad6A-ubiquitin complex bound to the nucleosome in two views. b Close-up view of ubiquitin and <t>H2B.</t> Two lysine residues (H2BK120 and H2BK116) near G76 of ubiquitin are shown. H2BS112, whose GlcNAcylation stimulates H2BK120 ubiquitination, is also shown. c Proposed mechanistic model. The wild-type Bre1 complex can bind to the nucleosome in two orientations, but H2BK120 ubiquitination occurs only when Bre1A binds to the acidic patch, as RING A , but not RING B , can recruit Rad6A and ubiquitin. Bre1B with G974T B -A978T B double substitution can recruit Rad6A and ubiquitin; thus, H2BK120 ubiquitination occurs in both binding modes.
    Anti Ubiquityl Histone H2b Lys120 D11 Xp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti h2bk120ub
    <t>H2BK120ub</t> is deficient in the Sertoli cells in the Amh-Rnf20 −/− mice. a Mass spectrometry detection of the ubiquitination of the H2B at K120 with the peptide HAVSEGTK(120)AVTK in the Rnf20 Flox/Flox . The MQ software was used to analyze the data from mass spectrometry. X axis, m/z; Y axis, the intensity of ions; y, the C-terminal fragment ion (Y series). b Ubiquitinated peptide information at the position of the K120 in the Rnf20 Flox/Flox . The ubiquitination modification site was not detected in the Amh-Rnf20 −/− . c Immunofluorescent analysis of SOX9, H2BK120ub, and DMRT1 on serial paraffin-sections in the Rnf20 Flox/Flox and the Amh-Rnf20 −/− testes at 7 days after birth and adult mice. The nuclei were stained with DAPI. TRITC signals represent the localization of H2BK120ub, while FITC signals showed the localization of SOX9 or DMRT1. The white squares in the panels correspond to the enlarged panels. Sn, Sertoli cells; Sg, spermatogonia. Scale bar, 10 μm
    Anti H2bk120ub, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti ubiquity histone h2b lys120
    <t>H2BK120ub</t> is deficient in the Sertoli cells in the Amh-Rnf20 −/− mice. a Mass spectrometry detection of the ubiquitination of the H2B at K120 with the peptide HAVSEGTK(120)AVTK in the Rnf20 Flox/Flox . The MQ software was used to analyze the data from mass spectrometry. X axis, m/z; Y axis, the intensity of ions; y, the C-terminal fragment ion (Y series). b Ubiquitinated peptide information at the position of the K120 in the Rnf20 Flox/Flox . The ubiquitination modification site was not detected in the Amh-Rnf20 −/− . c Immunofluorescent analysis of SOX9, H2BK120ub, and DMRT1 on serial paraffin-sections in the Rnf20 Flox/Flox and the Amh-Rnf20 −/− testes at 7 days after birth and adult mice. The nuclei were stained with DAPI. TRITC signals represent the localization of H2BK120ub, while FITC signals showed the localization of SOX9 or DMRT1. The white squares in the panels correspond to the enlarged panels. Sn, Sertoli cells; Sg, spermatogonia. Scale bar, 10 μm
    Anti Ubiquity Histone H2b Lys120, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc cat no 5546 rrid ab 10693452

    Cat No 5546 Rrid Ab 10693452, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti h2bub1 antibody
    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) <t>H2Bub1</t> and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).
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    Image Search Results


    a Model structure of the RINGA-RINGB-Rad6A-ubiquitin complex bound to the nucleosome in two views. b Close-up view of ubiquitin and H2B. Two lysine residues (H2BK120 and H2BK116) near G76 of ubiquitin are shown. H2BS112, whose GlcNAcylation stimulates H2BK120 ubiquitination, is also shown. c Proposed mechanistic model. The wild-type Bre1 complex can bind to the nucleosome in two orientations, but H2BK120 ubiquitination occurs only when Bre1A binds to the acidic patch, as RING A , but not RING B , can recruit Rad6A and ubiquitin. Bre1B with G974T B -A978T B double substitution can recruit Rad6A and ubiquitin; thus, H2BK120 ubiquitination occurs in both binding modes.

    Journal: Nature Communications

    Article Title: Structure of the human Bre1 complex bound to the nucleosome

    doi: 10.1038/s41467-024-46910-8

    Figure Lengend Snippet: a Model structure of the RINGA-RINGB-Rad6A-ubiquitin complex bound to the nucleosome in two views. b Close-up view of ubiquitin and H2B. Two lysine residues (H2BK120 and H2BK116) near G76 of ubiquitin are shown. H2BS112, whose GlcNAcylation stimulates H2BK120 ubiquitination, is also shown. c Proposed mechanistic model. The wild-type Bre1 complex can bind to the nucleosome in two orientations, but H2BK120 ubiquitination occurs only when Bre1A binds to the acidic patch, as RING A , but not RING B , can recruit Rad6A and ubiquitin. Bre1B with G974T B -A978T B double substitution can recruit Rad6A and ubiquitin; thus, H2BK120 ubiquitination occurs in both binding modes.

    Article Snippet: For Western blotting, Ubiquityl-Histone H2B (Lys120) (D11) Rabbit mAb (No. 5546; Cell Signaling) was used as the primary antibody (1:1000 dilution), goat anti-rabbit IgG-HRP (sc-2004; Santa Cruz Biotechnology) as the secondary antibody (1:2000 dilution), and ECL Prime (Cytiva) as the chemiluminescent reagent.

    Techniques: Binding Assay

    H2BK120ub is deficient in the Sertoli cells in the Amh-Rnf20 −/− mice. a Mass spectrometry detection of the ubiquitination of the H2B at K120 with the peptide HAVSEGTK(120)AVTK in the Rnf20 Flox/Flox . The MQ software was used to analyze the data from mass spectrometry. X axis, m/z; Y axis, the intensity of ions; y, the C-terminal fragment ion (Y series). b Ubiquitinated peptide information at the position of the K120 in the Rnf20 Flox/Flox . The ubiquitination modification site was not detected in the Amh-Rnf20 −/− . c Immunofluorescent analysis of SOX9, H2BK120ub, and DMRT1 on serial paraffin-sections in the Rnf20 Flox/Flox and the Amh-Rnf20 −/− testes at 7 days after birth and adult mice. The nuclei were stained with DAPI. TRITC signals represent the localization of H2BK120ub, while FITC signals showed the localization of SOX9 or DMRT1. The white squares in the panels correspond to the enlarged panels. Sn, Sertoli cells; Sg, spermatogonia. Scale bar, 10 μm

    Journal: Cell & Bioscience

    Article Title: RNF20 is required for male fertility through regulation of H2B ubiquitination in the Sertoli cells

    doi: 10.1186/s13578-023-01018-2

    Figure Lengend Snippet: H2BK120ub is deficient in the Sertoli cells in the Amh-Rnf20 −/− mice. a Mass spectrometry detection of the ubiquitination of the H2B at K120 with the peptide HAVSEGTK(120)AVTK in the Rnf20 Flox/Flox . The MQ software was used to analyze the data from mass spectrometry. X axis, m/z; Y axis, the intensity of ions; y, the C-terminal fragment ion (Y series). b Ubiquitinated peptide information at the position of the K120 in the Rnf20 Flox/Flox . The ubiquitination modification site was not detected in the Amh-Rnf20 −/− . c Immunofluorescent analysis of SOX9, H2BK120ub, and DMRT1 on serial paraffin-sections in the Rnf20 Flox/Flox and the Amh-Rnf20 −/− testes at 7 days after birth and adult mice. The nuclei were stained with DAPI. TRITC signals represent the localization of H2BK120ub, while FITC signals showed the localization of SOX9 or DMRT1. The white squares in the panels correspond to the enlarged panels. Sn, Sertoli cells; Sg, spermatogonia. Scale bar, 10 μm

    Article Snippet: The following primary antibodies were used: Anti-RNF20 (21625-1-AP, Proteintech Group, Rosemont, IL, USA), Anti-H2BK120ub (Cat# 5546s, Cell Signaling Technology, Danvers, MA, USA), Anti-β-ACTIN (Cat# 66009-1-Ig, Proteintech Group, Rosemont, IL, USA), Anti-SOX9 (Cat# 82,630 S, Cell Signaling Technology, Danvers, MA, USA), Anti-Caspase3 (Cat# 19677-1-AP, Proteintech Group, Rosemont, IL, USA), Anti-Claudin 11 (Cat# 36-4500, Thermo Fisher, Waltham, MA, USA), Anti-N-Cadherin (Cat# WL01047, Wanleibio, Shenyang, China), Anti-β-Catenin (Cat# 51067-2-AP, Proteintech Group, Rosemont, IL, USA), and Anti-α-Catenin (Cat# GTX111168, GeneTex, Texas, USA).

    Techniques: Mass Spectrometry, Software, Modification, Staining

    RNF20 deficiency in Sertoli cells impairs the Cldn11 transcription. a Scatterplots of differentially expressed genes. Red scatter, genes with significant up-regulated; blue scatter, genes with significant down-regulated; gray scatter, genes with no significant difference. X axis, Lg (WT FPKM) in the Rnf20 Flox/Flox ; Y axis, Lg (KO FPKM) in the Amh-Rnf20 −/− . b Gene ontology (GO) terms analysis of down-regulated genes in the Sertoli cells of the Amh-Rnf20 −/− testes. c, d Heatmaps showing the expression levels of down-regulated genes in the terms spermatogenesis ( c ) and cell adhesion ( d ) in the Sertoli cells of the Rnf20 Flox/Flox and the Amh-Rnf20 −/− . Color bar, Log 2 (FPKM). e Quantitative real-time PCR analysis of the genes Rnf20 and Cldn11 . The expression levels of the genes were related to Hprt expression. Relative levels, 2 −ΔCt ; T-tests were performed. *, p < 0.05, **, p < 0.01. f Western blot analysis of the expression levels of RNF20, CLDN11, and H2BK120ub proteins in adult mice. β-ACTIN was used as an internal control. g ChIP-PCR assays. The antibody specific for H2BK120ub was used in the ChIP analysis and primers were designed in the regions of promoter and exons of Cldn11 in the testes of the Rnf20 Flox/Flox and the Amh-Rnf20 −/− . The black graphs indicated the enriched levels in the Rnf20 Flox/Flox mice, while the white graphs indicated the levels in the Amh-Rnf20 −/− mice

    Journal: Cell & Bioscience

    Article Title: RNF20 is required for male fertility through regulation of H2B ubiquitination in the Sertoli cells

    doi: 10.1186/s13578-023-01018-2

    Figure Lengend Snippet: RNF20 deficiency in Sertoli cells impairs the Cldn11 transcription. a Scatterplots of differentially expressed genes. Red scatter, genes with significant up-regulated; blue scatter, genes with significant down-regulated; gray scatter, genes with no significant difference. X axis, Lg (WT FPKM) in the Rnf20 Flox/Flox ; Y axis, Lg (KO FPKM) in the Amh-Rnf20 −/− . b Gene ontology (GO) terms analysis of down-regulated genes in the Sertoli cells of the Amh-Rnf20 −/− testes. c, d Heatmaps showing the expression levels of down-regulated genes in the terms spermatogenesis ( c ) and cell adhesion ( d ) in the Sertoli cells of the Rnf20 Flox/Flox and the Amh-Rnf20 −/− . Color bar, Log 2 (FPKM). e Quantitative real-time PCR analysis of the genes Rnf20 and Cldn11 . The expression levels of the genes were related to Hprt expression. Relative levels, 2 −ΔCt ; T-tests were performed. *, p < 0.05, **, p < 0.01. f Western blot analysis of the expression levels of RNF20, CLDN11, and H2BK120ub proteins in adult mice. β-ACTIN was used as an internal control. g ChIP-PCR assays. The antibody specific for H2BK120ub was used in the ChIP analysis and primers were designed in the regions of promoter and exons of Cldn11 in the testes of the Rnf20 Flox/Flox and the Amh-Rnf20 −/− . The black graphs indicated the enriched levels in the Rnf20 Flox/Flox mice, while the white graphs indicated the levels in the Amh-Rnf20 −/− mice

    Article Snippet: The following primary antibodies were used: Anti-RNF20 (21625-1-AP, Proteintech Group, Rosemont, IL, USA), Anti-H2BK120ub (Cat# 5546s, Cell Signaling Technology, Danvers, MA, USA), Anti-β-ACTIN (Cat# 66009-1-Ig, Proteintech Group, Rosemont, IL, USA), Anti-SOX9 (Cat# 82,630 S, Cell Signaling Technology, Danvers, MA, USA), Anti-Caspase3 (Cat# 19677-1-AP, Proteintech Group, Rosemont, IL, USA), Anti-Claudin 11 (Cat# 36-4500, Thermo Fisher, Waltham, MA, USA), Anti-N-Cadherin (Cat# WL01047, Wanleibio, Shenyang, China), Anti-β-Catenin (Cat# 51067-2-AP, Proteintech Group, Rosemont, IL, USA), and Anti-α-Catenin (Cat# GTX111168, GeneTex, Texas, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Journal: Cell

    Article Title: Recycling of modified H2A-H2B provides short-term memory of chromatin states

    doi: 10.1016/j.cell.2023.01.007

    Figure Lengend Snippet:

    Article Snippet: H2BK120ub (rabbit) , Cell Signaling Technology , Cat no. 5546; RRID: AB_10693452.

    Techniques: Recombinant, Purification, Western Blot, Software

    The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    doi: 10.1016/j.omtn.2023.02.014

    Figure Lengend Snippet: The core circadian component BMAL1 regulated histone H2B monoubiquitination levels (A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log 2 FC and −log 10 (q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log 2 FC and −log 10 (q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).

    Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

    Techniques: RNA Sequencing Assay, Infection, Expressing

    BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    doi: 10.1016/j.omtn.2023.02.014

    Figure Lengend Snippet: BMAL1 targeted the circadian-controlled gene TTK to regulate H2Bub1 levels to affect the osteogenic capacity of MSCs (A and B) Relative mRNA (A) and protein (B) expression of TTK in the MSCs infected with Sh-NC, Sh-BMAL1, OE-NC, or OE-BMAL1 lentiviruses on the 10th day of osteogenic differentiation. (C) The putative E-boxes in the TTK promoter region. (D) CUT&Tag-qPCR showed the percentage of BMAL1 occupancy on the TTK promoter. Data are shown as the proportion of input level and normalized to the IgG control. (E) CUT&Tag-qPCR analysis showing the H2Bub1 occupancy on RUNX2 and OSX in the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. (F and G) Relative mRNA expression (F) and protein expression (G) of RUNX2 and OSX in MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses that were undergoing osteogenic differentiation. Bar graphs showing the relative expression. (H) ARS and ALP staining of the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses on the 14th day of osteogenic differentiation. (I) HE and Masson staining and Col I immunohistochemistry of transplanted HA/TCP embedded with the MSCs infected with Sh-NC and OE-NC, Sh-BMAL1 and OE-NC, or Sh-BMAL1 and OE-TTK lentiviruses. All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

    Techniques: Expressing, Infection, Staining, Immunohistochemistry

    H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    doi: 10.1016/j.omtn.2023.02.014

    Figure Lengend Snippet: H2Bub1 positively modulated the expression of BMAL1 at the transcript level (A) Signal traces of ChIP-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in hFOB1.19 cells on day 0 or 7 of osteogenic differentiation. (B and C) Relative mRNA expression (B) and protein expression (C) of BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. Bar graphs showing the relative expression. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (D) CUT&Tag-seq average binding profiles and heatmaps depicting occupancy of H2Bub1 and Pol II in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. (E) GO biological process analyses of the CUT&Tag-seq data comparing between the Sh-NC and Sh-RNF40 groups and the Sh-NC and Sh-WAC groups. Bar graph showing the p values of the enriched terms. (F) Signal traces of CUT&Tag-seq data showing H2Bub1 and Pol II occupancy on BMAL1 in the MSCs infected with Sh-NC, Sh-RNF40, or Sh-WAC lentiviruses. The colorful shadows showing regions with difference (G) CUT&Tag-qPCR analysis showing the H2Bub1 and Pol II occupancy on BMAL1 sites A–F in the MSCs infected with Sh-NC, Sh-RNF40 or Sh-WAC lentiviruses. Data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

    Techniques: Expressing, ChIP-sequencing, Infection, Binding Assay

    TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    doi: 10.1016/j.omtn.2023.02.014

    Figure Lengend Snippet: TTK expression and H2Bub1 levels were decreased in BM-MSCs in senile osteoporosis (A and B) Western blot analysis of the levels of TTK and H2Bub1 in BM-MSCs from 2-month-old and 20-month-old mice, patients with traffic injuries and patients with senile osteoporosis. (C and D) Immunofluorescence staining (scale bar, 100 μm) showed Ttk expression and H2Bub1 levels in the Ocn + osteoblast lineage in 2-month-old and 20-month-old mice (white arrows). (E and F) Immunofluorescence staining (scale bar, 100 μm) showed TTK expression and H2Bub1 levels in the OCN + osteoblast lineage in young patients with traffic injuries and patients with senile osteoporosis (white arrows). All data are presented as mean ± SD; n = 3; ∗p < 0.05.

    Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

    Techniques: Expressing, Western Blot, Immunofluorescence, Staining

    Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    doi: 10.1016/j.omtn.2023.02.014

    Figure Lengend Snippet: Bone-targeted Bmal1 or Ttk rescue-treated senile osteoporosis (A) Diagram showing the workflow of rAAV9 injection in 18-month-old mice with calvarial and femoral defects and bone section analysis. (B) Immunoblot analysis showing mNeonGreen expression in different organs of the mice injected with rAAV9. (C) Fluorescence images of different organs of mice injected with rAAV9. (D) Immunofluorescence staining (scale bar, 100 μm) showing mNeonGreen-expressing osteoblasts in the femurs of the mice injected with rAAV9. (E) Immunofluorescence staining (scale bar, 100 μm) showing Bmal1 expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control or rAAV9-Bmal1 (white arrows). (F) Immunofluorescence staining (scale bar, 100 μm) showing Ttk expression in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). (G) Immunofluorescence staining (scale bar, 100 μm) showing H2Bub1 levels in the Ocn + osteoblast lineage in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk (white arrows). Data are presented as mean ± SD; n = 3; ∗p < 0.05. (H) Micro-CT analysis comparing the healing rates of calvarial and femoral defects in the mice injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. (I) Representative micro-CT images showing the trabecular bone of mice with senile osteoporosis injected with rAAV9-control, rAAV9-Bmal1, or rAAV9-Ttk. Bone morphometric analysis, including the analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Ct.Th., was performed. Data are presented as mean ± SD; n = 5; ∗p < 0.05.

    Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

    Techniques: Injection, Western Blot, Expressing, Fluorescence, Immunofluorescence, Staining, Micro-CT

    Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis

    doi: 10.1016/j.omtn.2023.02.014

    Figure Lengend Snippet: Model showing that the disruption of the BMAL1-TTK-MDM2-H2Bub1 positive loop led to the impaired osteogenic capacity of BM-MSCs in senile osteoporosis

    Article Snippet: The following primary antibodies were used: anti-BMAL1 antibody (catalog no. 14020; Cell Signaling Technology), anti-TTK antibody (catalog no. ab11108; Abcam), anti-CLOCK antibody (catalog no. ab3517; Abcam), anti-OCN antibody (catalog no. 29560; Sab), anti-GAPDH (catalog no. 5174S; Cell Signaling Technology), anti-RUNX2 (catalog no. 12556S; Cell Signaling Technology), anti-OSX (catalog no. ab209484; Abcam), anti-β-tubulin (catalog no. 2128; Cell Signaling Technology), anti-OPN antibody (catalog no. 42036; Sab), anti-RNF20 antibody (catalog no. ab181104; Abcam), anti-RNF40 antibody (catalog no. ab191309; Abcam), anti-WAC antibody (catalog no. ab109486; Abcam), anti-H2B antibody (catalog no. 12364; Cell Signaling Technology), anti-H2Bub1 antibody (catalog no. 5546; Cell Signaling Technology), anti-H2A antibody (catalog no. 12349; Cell Signaling Technology) anti-H2Aub1 antibody (catalog no. 8240; Cell Signaling Technology), anti-MDM2 antibody (catalog no. ab226939; Abcam), and anti-pan phosphoserine/threonine antibody (catalog no. AP1067; Abclonal).

    Techniques: