rabbit polyclonal anti hp1γ cbx3 ab Search Results


anti hp1γ antibody  (Proteintech)
93
Proteintech anti hp1γ antibody
Anti Hp1γ Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+anti+hp1%CE%B3+cbx3+ab/CBX3+Antibody/pmc07199406-615-28-31
Average 93 stars, based on 1 article reviews
anti hp1γ antibody - by Bioz Stars, 2026-10
93/100 stars
  Buy from Supplier
anti targeting hp1γ  (ABclonal Biotechnology)
93
ABclonal Biotechnology anti targeting hp1γ
Anti Targeting Hp1γ, supplied by ABclonal Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+anti+hp1%CE%B3+cbx3+ab/%5BKO+Validated%5D+CBX3+Rabbit+pAb/pm41223182-102-22-27
Average 93 stars, based on 1 article reviews
anti targeting hp1γ - by Bioz Stars, 2026-10
93/100 stars
  Buy from Supplier
rabbit polyclonal anti hp1γ cbx3 ab  (Cell Signaling Technology Inc)
94
Cell Signaling Technology Inc rabbit polyclonal anti hp1γ cbx3 ab
Figure 1. Identification of MC antigens reactive to IgA auto-Abs generated in gddY mice. (A) Detection of antigens for rmAbs derived from gddY mouse PBs in the lysates of gddY mouse primary MCs by WB. Split membranes, each containing proteins in one lane, were incubated with the indicated rmAbs (1 μg/ml), followed by an anti-human IgG Ab. (B) Immunoprecipitation of gddY mouse MC lysates with rmAb#66 or the control rmAb#NP. SDS–PAGE of the precipitates was developed using silver staining. The bands indicated by arrows were excised and analyzed using mass spectrometry. Those from the rmAb#66 precipitate included CBX1, <t>CBX3,</t> and CBX5, as indicated by arrows. (C) FLAG-tagged CBX1, CBX3, or CBX5 proteins transiently expressed in HEK293T cells were detected by WB with the indicated rmAbs (1 μg/ml), followed by anti-human IgG Ab (left panel) or anti-FLAG Ab (right panel). (D) ELISA screening of rmAbs for binding to CBX proteins. The heatmap represents the resulting OD values of the indicated rmAbs, with “+” indicating a saturated signal. (E) Reactivity of gddY mouse serum IgA with the CBX proteins. WB was performed as in (C) using sera of individual BALB/c or gddY mice as the primary Ab as indicated and anti-IgA Ab as the secondary Ab (left, top) or anti-FLAG Ab to confirm the presence of CBX proteins on the blot (left, bottom). Representative data are shown here. Venn diagram showing the percentage of gddY mice whose serum IgA reacted with CBX1, CBX3, or CBX5 (right panel) (n = 14). (F) ELISA determination of anti-CBX3 IgA Abs in sera from healthy controls (HC; n = 30) and patients with IgAN (IgAN; n = 70). ***: 0.0006 (Mann–Whitney U test). The dashed line shows 99% confidence interval (CI) of the control sera. One of two or three independent experiments with similar results is shown in each panel. Source data are available for this figure.
Rabbit Polyclonal Anti Hp1γ Cbx3 Ab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+anti+hp1%CE%B3+cbx3+ab/HP1gamma+Antibody/pm38331476-205-34-40
Average 94 stars, based on 1 article reviews
rabbit polyclonal anti hp1γ cbx3 ab - by Bioz Stars, 2026-10
94/100 stars
  Buy from Supplier
cbx3  (Santa Cruz Biotechnology)
94
Santa Cruz Biotechnology cbx3
( A ) FLAG-tagged <t>CBX3</t> was immunoprecipitated from 293T cells and subjected to MS analysis to identify CBX3 interacting proteins. ( B ) The list of top hits proteins from CBX3 MS. ( C ) Western blot (WB) analysis of whole-cell lysates (WCL) and anti-CBX3 immunoprecipitations (IPs) derived from PC-3 cells. ( D ) WB analysis of WCL and anti-RPA2 IPs derived from PC-3 cells. ( E ) Colocalization of CBX3 with RPA2 foci after noted drug treatment or transfected with indicated small interfering RNA (siRNA) in U2OS cells. Scale bar, 10 μm. ( F ) Quantitation of average CBX3 foci number in each cell. Data were presented as means ± SD of more than 200 cells from three biological replicates. n.s., not significant. Two-tailed unpaired Student’s t test, *** P < 0.001. ( G ) Replication fork-bound proteins were isolated by the iPOND assay with control and RPA2 knockdown U2OS cells. ( H ) Schematic diagram depicting a set of GST-RPA2 recombinant protein constructs. ( I ) WB analysis of Flag-CBX3 proteins in U2OS cells pulled down by GST or GST-RPA2 recombinant proteins. ( J ) Schematic diagram depicting a set of GST-CBX3 recombinant protein constructs. ( K ) WB of His-RPA2 proteins in U2OS cells pulled down by GST or GST-CBX3 recombinant proteins. ( L and M ) U2OS cells transfected with Flag-CBX3 proteins were subjected to immunofluorescence staining (IFC) after HU treatment. Representative images were shown in (L) and quantitative data are shown in (M). Scale bar, 10 μm. Data were shown as the mean ± SD from three biological replicates, two-tailed unpaired Student’s t test, *** P < 0.001. IgG, immunoglobulin.
Cbx3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+anti+hp1%CE%B3+cbx3+ab/HP1%CE%B3+Antibody/pmc11114232-172-4-6
Average 94 stars, based on 1 article reviews
cbx3 - by Bioz Stars, 2026-10
94/100 stars
  Buy from Supplier
hp1γ cbx3 immunolabeling  (Bethyl)
91
Bethyl hp1γ cbx3 immunolabeling
Persistent hyperemic foci exhibit evidence of dermal senescence. ( A – E ) Dermal cells in areas of high Hgb content exhibit a heterochromatin pattern of DAPI staining. After excision of skin from mice treated with and without UV at 2 and 20 weeks after stopping UV treatments, the sections were stained with DAPI to better demonstrate nuclear morphology. Nuclei of dermal cells showing a heterochromatin staining pattern are shown by white arrows while nuclei exhibiting a euchromatin pattern of DAPI staining are shown by yellow arrows. The hatched white line outlines the epidermal-dermal junction. The white scale bar represents 50 µm. ( A ) Skin from an area of low Hgb content 2 weeks after stopping UV treatments. ( B ) Skin from an area of high Hgb content 2 weeks after stopping UV treatments. ( C ) Control non-UV treated skin. ( D ) Skin from an area of low Hgb content 20 weeks after stopping UV. ( E ) Skin from an area of high Hgb content 20 weeks after stopping UV. (F – J ) Representative photomigrographs of skin following <t>immunolabeling</t> with anti-p16 INK4a antibodies. ( F ) Control non-UV treated epidermis. ( G , H ) Skin excised 2 weeks after stopping UV treatments from an area of low Hgb content ( G ) or from an area of high Hgb content ( H ). ( I , J ) Skin excised from a low Hgb area ( I ) or a high Hgb area ( J ) at 20 weeks after stopping UV treatments. Black arrows in ( H , J ) show dermal cells with enlarged nuclei labeling positive for p16 INK4a . The black scale bars represent 100 µm. ( K ) <t>HP1γ</t> + dermal cells are increased in hyperemic areas at both 2 and 20 weeks after stopping UV treatments. Immunofluorescent (IF) labeling of formalin-fixed skin sections was performed using anti-HP1γ and anti-pancytokeratin (CK) antibody. The data depicts the % of dermal cells positive for HP1γ nuclear labeling. ( L ) Dermal cells positive for nuclear γH2AX are also increased in hyperemic areas only at 2 and 20 weeks post-UV. IF was performed for both nuclear γH2AX immunolabeling and CK. The data shown is the percentage of γH2AX + dermal cells relative to all CK negative cells. (ns = non-significant; ** = p < 0.01; *** = p < 0.001).
Hp1γ Cbx3 Immunolabeling, supplied by Bethyl, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+anti+hp1%CE%B3+cbx3+ab/CBX3+Antibody/pmc07645611-200-17-27
Average 91 stars, based on 1 article reviews
hp1γ cbx3 immunolabeling - by Bioz Stars, 2026-10
91/100 stars
  Buy from Supplier
rabbit anti hp1γ  (Novus Biologicals)
93
Novus Biologicals rabbit anti hp1γ
Persistent hyperemic foci exhibit evidence of dermal senescence. ( A – E ) Dermal cells in areas of high Hgb content exhibit a heterochromatin pattern of DAPI staining. After excision of skin from mice treated with and without UV at 2 and 20 weeks after stopping UV treatments, the sections were stained with DAPI to better demonstrate nuclear morphology. Nuclei of dermal cells showing a heterochromatin staining pattern are shown by white arrows while nuclei exhibiting a euchromatin pattern of DAPI staining are shown by yellow arrows. The hatched white line outlines the epidermal-dermal junction. The white scale bar represents 50 µm. ( A ) Skin from an area of low Hgb content 2 weeks after stopping UV treatments. ( B ) Skin from an area of high Hgb content 2 weeks after stopping UV treatments. ( C ) Control non-UV treated skin. ( D ) Skin from an area of low Hgb content 20 weeks after stopping UV. ( E ) Skin from an area of high Hgb content 20 weeks after stopping UV. (F – J ) Representative photomigrographs of skin following <t>immunolabeling</t> with anti-p16 INK4a antibodies. ( F ) Control non-UV treated epidermis. ( G , H ) Skin excised 2 weeks after stopping UV treatments from an area of low Hgb content ( G ) or from an area of high Hgb content ( H ). ( I , J ) Skin excised from a low Hgb area ( I ) or a high Hgb area ( J ) at 20 weeks after stopping UV treatments. Black arrows in ( H , J ) show dermal cells with enlarged nuclei labeling positive for p16 INK4a . The black scale bars represent 100 µm. ( K ) <t>HP1γ</t> + dermal cells are increased in hyperemic areas at both 2 and 20 weeks after stopping UV treatments. Immunofluorescent (IF) labeling of formalin-fixed skin sections was performed using anti-HP1γ and anti-pancytokeratin (CK) antibody. The data depicts the % of dermal cells positive for HP1γ nuclear labeling. ( L ) Dermal cells positive for nuclear γH2AX are also increased in hyperemic areas only at 2 and 20 weeks post-UV. IF was performed for both nuclear γH2AX immunolabeling and CK. The data shown is the percentage of γH2AX + dermal cells relative to all CK negative cells. (ns = non-significant; ** = p < 0.01; *** = p < 0.001).
Rabbit Anti Hp1γ, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+anti+hp1%CE%B3+cbx3+ab/CBX3+Antibody/pmc12168070-52-54-57
Average 93 stars, based on 1 article reviews
rabbit anti hp1γ - by Bioz Stars, 2026-10
93/100 stars
  Buy from Supplier
rabbit anti cbx3  (Bethyl)
91
Bethyl rabbit anti cbx3
Persistent hyperemic foci exhibit evidence of dermal senescence. ( A – E ) Dermal cells in areas of high Hgb content exhibit a heterochromatin pattern of DAPI staining. After excision of skin from mice treated with and without UV at 2 and 20 weeks after stopping UV treatments, the sections were stained with DAPI to better demonstrate nuclear morphology. Nuclei of dermal cells showing a heterochromatin staining pattern are shown by white arrows while nuclei exhibiting a euchromatin pattern of DAPI staining are shown by yellow arrows. The hatched white line outlines the epidermal-dermal junction. The white scale bar represents 50 µm. ( A ) Skin from an area of low Hgb content 2 weeks after stopping UV treatments. ( B ) Skin from an area of high Hgb content 2 weeks after stopping UV treatments. ( C ) Control non-UV treated skin. ( D ) Skin from an area of low Hgb content 20 weeks after stopping UV. ( E ) Skin from an area of high Hgb content 20 weeks after stopping UV. (F – J ) Representative photomigrographs of skin following <t>immunolabeling</t> with anti-p16 INK4a antibodies. ( F ) Control non-UV treated epidermis. ( G , H ) Skin excised 2 weeks after stopping UV treatments from an area of low Hgb content ( G ) or from an area of high Hgb content ( H ). ( I , J ) Skin excised from a low Hgb area ( I ) or a high Hgb area ( J ) at 20 weeks after stopping UV treatments. Black arrows in ( H , J ) show dermal cells with enlarged nuclei labeling positive for p16 INK4a . The black scale bars represent 100 µm. ( K ) <t>HP1γ</t> + dermal cells are increased in hyperemic areas at both 2 and 20 weeks after stopping UV treatments. Immunofluorescent (IF) labeling of formalin-fixed skin sections was performed using anti-HP1γ and anti-pancytokeratin (CK) antibody. The data depicts the % of dermal cells positive for HP1γ nuclear labeling. ( L ) Dermal cells positive for nuclear γH2AX are also increased in hyperemic areas only at 2 and 20 weeks post-UV. IF was performed for both nuclear γH2AX immunolabeling and CK. The data shown is the percentage of γH2AX + dermal cells relative to all CK negative cells. (ns = non-significant; ** = p < 0.01; *** = p < 0.001).
Rabbit Anti Cbx3, supplied by Bethyl, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+anti+hp1%CE%B3+cbx3+ab/CBX3+IHC+Antibody/pmc07645611-200-23-27
Average 91 stars, based on 1 article reviews
rabbit anti cbx3 - by Bioz Stars, 2026-10
91/100 stars
  Buy from Supplier
monoclonal rabbit anti γh2ax  (Bethyl)
93
Bethyl monoclonal rabbit anti γh2ax
Persistent hyperemic foci exhibit evidence of dermal senescence. ( A – E ) Dermal cells in areas of high Hgb content exhibit a heterochromatin pattern of DAPI staining. After excision of skin from mice treated with and without UV at 2 and 20 weeks after stopping UV treatments, the sections were stained with DAPI to better demonstrate nuclear morphology. Nuclei of dermal cells showing a heterochromatin staining pattern are shown by white arrows while nuclei exhibiting a euchromatin pattern of DAPI staining are shown by yellow arrows. The hatched white line outlines the epidermal-dermal junction. The white scale bar represents 50 µm. ( A ) Skin from an area of low Hgb content 2 weeks after stopping UV treatments. ( B ) Skin from an area of high Hgb content 2 weeks after stopping UV treatments. ( C ) Control non-UV treated skin. ( D ) Skin from an area of low Hgb content 20 weeks after stopping UV. ( E ) Skin from an area of high Hgb content 20 weeks after stopping UV. (F – J ) Representative photomigrographs of skin following immunolabeling with anti-p16 INK4a antibodies. ( F ) Control non-UV treated epidermis. ( G , H ) Skin excised 2 weeks after stopping UV treatments from an area of low Hgb content ( G ) or from an area of high Hgb content ( H ). ( I , J ) Skin excised from a low Hgb area ( I ) or a high Hgb area ( J ) at 20 weeks after stopping UV treatments. Black arrows in ( H , J ) show dermal cells with enlarged nuclei labeling positive for p16 INK4a . The black scale bars represent 100 µm. ( K ) HP1γ + dermal cells are increased in hyperemic areas at both 2 and 20 weeks after stopping UV treatments. Immunofluorescent (IF) labeling of formalin-fixed skin sections was performed using anti-HP1γ and anti-pancytokeratin (CK) antibody. The data depicts the % of dermal cells positive for HP1γ nuclear labeling. ( L ) Dermal cells positive for nuclear <t>γH2AX</t> are also increased in hyperemic areas only at 2 and 20 weeks post-UV. IF was performed for both nuclear γH2AX immunolabeling and CK. The data shown is the percentage of γH2AX + dermal cells relative to all CK negative cells. (ns = non-significant; ** = p < 0.01; *** = p < 0.001).
Monoclonal Rabbit Anti γh2ax, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+anti+hp1%CE%B3+cbx3+ab/gamma-H2AX+Recombinant+Monoclonal+Antibody/pmc07645611-200-34-38
Average 93 stars, based on 1 article reviews
monoclonal rabbit anti γh2ax - by Bioz Stars, 2026-10
93/100 stars
  Buy from Supplier
rabbit igg  (Jackson Immuno)
95
Jackson Immuno rabbit igg
Persistent hyperemic foci exhibit evidence of dermal senescence. ( A – E ) Dermal cells in areas of high Hgb content exhibit a heterochromatin pattern of DAPI staining. After excision of skin from mice treated with and without UV at 2 and 20 weeks after stopping UV treatments, the sections were stained with DAPI to better demonstrate nuclear morphology. Nuclei of dermal cells showing a heterochromatin staining pattern are shown by white arrows while nuclei exhibiting a euchromatin pattern of DAPI staining are shown by yellow arrows. The hatched white line outlines the epidermal-dermal junction. The white scale bar represents 50 µm. ( A ) Skin from an area of low Hgb content 2 weeks after stopping UV treatments. ( B ) Skin from an area of high Hgb content 2 weeks after stopping UV treatments. ( C ) Control non-UV treated skin. ( D ) Skin from an area of low Hgb content 20 weeks after stopping UV. ( E ) Skin from an area of high Hgb content 20 weeks after stopping UV. (F – J ) Representative photomigrographs of skin following immunolabeling with anti-p16 INK4a antibodies. ( F ) Control non-UV treated epidermis. ( G , H ) Skin excised 2 weeks after stopping UV treatments from an area of low Hgb content ( G ) or from an area of high Hgb content ( H ). ( I , J ) Skin excised from a low Hgb area ( I ) or a high Hgb area ( J ) at 20 weeks after stopping UV treatments. Black arrows in ( H , J ) show dermal cells with enlarged nuclei labeling positive for p16 INK4a . The black scale bars represent 100 µm. ( K ) HP1γ + dermal cells are increased in hyperemic areas at both 2 and 20 weeks after stopping UV treatments. Immunofluorescent (IF) labeling of formalin-fixed skin sections was performed using anti-HP1γ and anti-pancytokeratin (CK) antibody. The data depicts the % of dermal cells positive for HP1γ nuclear labeling. ( L ) Dermal cells positive for nuclear <t>γH2AX</t> are also increased in hyperemic areas only at 2 and 20 weeks post-UV. IF was performed for both nuclear γH2AX immunolabeling and CK. The data shown is the percentage of γH2AX + dermal cells relative to all CK negative cells. (ns = non-significant; ** = p < 0.01; *** = p < 0.001).
Rabbit Igg, supplied by Jackson Immuno, 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/product/rabbit+polyclonal+anti+hp1%CE%B3+cbx3+ab/ChromPure+Rabbit+IgG%2C+whole+molecule/pm38331476-205-52-58
Average 95 stars, based on 1 article reviews
rabbit igg - by Bioz Stars, 2026-10
95/100 stars
  Buy from Supplier
anti mouse iga apc  (SouthernBiotech)
93
SouthernBiotech anti mouse iga apc
Persistent hyperemic foci exhibit evidence of dermal senescence. ( A – E ) Dermal cells in areas of high Hgb content exhibit a heterochromatin pattern of DAPI staining. After excision of skin from mice treated with and without UV at 2 and 20 weeks after stopping UV treatments, the sections were stained with DAPI to better demonstrate nuclear morphology. Nuclei of dermal cells showing a heterochromatin staining pattern are shown by white arrows while nuclei exhibiting a euchromatin pattern of DAPI staining are shown by yellow arrows. The hatched white line outlines the epidermal-dermal junction. The white scale bar represents 50 µm. ( A ) Skin from an area of low Hgb content 2 weeks after stopping UV treatments. ( B ) Skin from an area of high Hgb content 2 weeks after stopping UV treatments. ( C ) Control non-UV treated skin. ( D ) Skin from an area of low Hgb content 20 weeks after stopping UV. ( E ) Skin from an area of high Hgb content 20 weeks after stopping UV. (F – J ) Representative photomigrographs of skin following immunolabeling with anti-p16 INK4a antibodies. ( F ) Control non-UV treated epidermis. ( G , H ) Skin excised 2 weeks after stopping UV treatments from an area of low Hgb content ( G ) or from an area of high Hgb content ( H ). ( I , J ) Skin excised from a low Hgb area ( I ) or a high Hgb area ( J ) at 20 weeks after stopping UV treatments. Black arrows in ( H , J ) show dermal cells with enlarged nuclei labeling positive for p16 INK4a . The black scale bars represent 100 µm. ( K ) HP1γ + dermal cells are increased in hyperemic areas at both 2 and 20 weeks after stopping UV treatments. Immunofluorescent (IF) labeling of formalin-fixed skin sections was performed using anti-HP1γ and anti-pancytokeratin (CK) antibody. The data depicts the % of dermal cells positive for HP1γ nuclear labeling. ( L ) Dermal cells positive for nuclear <t>γH2AX</t> are also increased in hyperemic areas only at 2 and 20 weeks post-UV. IF was performed for both nuclear γH2AX immunolabeling and CK. The data shown is the percentage of γH2AX + dermal cells relative to all CK negative cells. (ns = non-significant; ** = p < 0.01; *** = p < 0.001).
Anti Mouse Iga Apc, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+anti+hp1%CE%B3+cbx3+ab/Rat+Anti-Mouse+IgA-APC/pmc10853438-235-7-11
Average 93 stars, based on 1 article reviews
anti mouse iga apc - by Bioz Stars, 2026-10
93/100 stars
  Buy from Supplier
anti human igg fcγ alexa fluor 647  (Jackson Immuno)
93
Jackson Immuno anti human igg fcγ alexa fluor 647
Persistent hyperemic foci exhibit evidence of dermal senescence. ( A – E ) Dermal cells in areas of high Hgb content exhibit a heterochromatin pattern of DAPI staining. After excision of skin from mice treated with and without UV at 2 and 20 weeks after stopping UV treatments, the sections were stained with DAPI to better demonstrate nuclear morphology. Nuclei of dermal cells showing a heterochromatin staining pattern are shown by white arrows while nuclei exhibiting a euchromatin pattern of DAPI staining are shown by yellow arrows. The hatched white line outlines the epidermal-dermal junction. The white scale bar represents 50 µm. ( A ) Skin from an area of low Hgb content 2 weeks after stopping UV treatments. ( B ) Skin from an area of high Hgb content 2 weeks after stopping UV treatments. ( C ) Control non-UV treated skin. ( D ) Skin from an area of low Hgb content 20 weeks after stopping UV. ( E ) Skin from an area of high Hgb content 20 weeks after stopping UV. (F – J ) Representative photomigrographs of skin following immunolabeling with anti-p16 INK4a antibodies. ( F ) Control non-UV treated epidermis. ( G , H ) Skin excised 2 weeks after stopping UV treatments from an area of low Hgb content ( G ) or from an area of high Hgb content ( H ). ( I , J ) Skin excised from a low Hgb area ( I ) or a high Hgb area ( J ) at 20 weeks after stopping UV treatments. Black arrows in ( H , J ) show dermal cells with enlarged nuclei labeling positive for p16 INK4a . The black scale bars represent 100 µm. ( K ) HP1γ + dermal cells are increased in hyperemic areas at both 2 and 20 weeks after stopping UV treatments. Immunofluorescent (IF) labeling of formalin-fixed skin sections was performed using anti-HP1γ and anti-pancytokeratin (CK) antibody. The data depicts the % of dermal cells positive for HP1γ nuclear labeling. ( L ) Dermal cells positive for nuclear <t>γH2AX</t> are also increased in hyperemic areas only at 2 and 20 weeks post-UV. IF was performed for both nuclear γH2AX immunolabeling and CK. The data shown is the percentage of γH2AX + dermal cells relative to all CK negative cells. (ns = non-significant; ** = p < 0.01; *** = p < 0.001).
Anti Human Igg Fcγ Alexa Fluor 647, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+anti+hp1%CE%B3+cbx3+ab/Alexa+Fluor+647+AffiniPure+F(ab')%E2%82%82+Fragment+Donkey+Anti-Human+IgG%2C+Fc%CE%B3+fragment+specific/pmc10853438-235-65-71
Average 93 stars, based on 1 article reviews
anti human igg fcγ alexa fluor 647 - by Bioz Stars, 2026-10
93/100 stars
  Buy from Supplier
rabbit monoclonal anti hp1β cbx1 ab  (Cell Signaling Technology Inc)
93
Cell Signaling Technology Inc rabbit monoclonal anti hp1β cbx1 ab
Figure 1. Identification of MC antigens reactive to IgA auto-Abs generated in gddY mice. (A) Detection of antigens for rmAbs derived from gddY mouse PBs in the lysates of gddY mouse primary MCs by WB. Split membranes, each containing proteins in one lane, were incubated with the indicated rmAbs (1 μg/ml), followed by an anti-human IgG Ab. (B) Immunoprecipitation of gddY mouse MC lysates with rmAb#66 or the control rmAb#NP. SDS–PAGE of the precipitates was developed using silver staining. The bands indicated by arrows were excised and analyzed using mass spectrometry. Those from the rmAb#66 precipitate included <t>CBX1,</t> CBX3, and CBX5, as indicated by arrows. (C) FLAG-tagged CBX1, CBX3, or CBX5 proteins transiently expressed in HEK293T cells were detected by WB with the indicated rmAbs (1 μg/ml), followed by anti-human IgG Ab (left panel) or anti-FLAG Ab (right panel). (D) ELISA screening of rmAbs for binding to CBX proteins. The heatmap represents the resulting OD values of the indicated rmAbs, with “+” indicating a saturated signal. (E) Reactivity of gddY mouse serum IgA with the CBX proteins. WB was performed as in (C) using sera of individual BALB/c or gddY mice as the primary Ab as indicated and anti-IgA Ab as the secondary Ab (left, top) or anti-FLAG Ab to confirm the presence of CBX proteins on the blot (left, bottom). Representative data are shown here. Venn diagram showing the percentage of gddY mice whose serum IgA reacted with CBX1, CBX3, or CBX5 (right panel) (n = 14). (F) ELISA determination of anti-CBX3 IgA Abs in sera from healthy controls (HC; n = 30) and patients with IgAN (IgAN; n = 70). ***: 0.0006 (Mann–Whitney U test). The dashed line shows 99% confidence interval (CI) of the control sera. One of two or three independent experiments with similar results is shown in each panel. Source data are available for this figure.
Rabbit Monoclonal Anti Hp1β Cbx1 Ab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+anti+hp1%CE%B3+cbx3+ab/HP1beta+XP+Rabbit+mAb/pm38331476-205-43-49
Average 93 stars, based on 1 article reviews
rabbit monoclonal anti hp1β cbx1 ab - by Bioz Stars, 2026-10
93/100 stars
  Buy from Supplier

Image Search Results


Figure 1. Identification of MC antigens reactive to IgA auto-Abs generated in gddY mice. (A) Detection of antigens for rmAbs derived from gddY mouse PBs in the lysates of gddY mouse primary MCs by WB. Split membranes, each containing proteins in one lane, were incubated with the indicated rmAbs (1 μg/ml), followed by an anti-human IgG Ab. (B) Immunoprecipitation of gddY mouse MC lysates with rmAb#66 or the control rmAb#NP. SDS–PAGE of the precipitates was developed using silver staining. The bands indicated by arrows were excised and analyzed using mass spectrometry. Those from the rmAb#66 precipitate included CBX1, CBX3, and CBX5, as indicated by arrows. (C) FLAG-tagged CBX1, CBX3, or CBX5 proteins transiently expressed in HEK293T cells were detected by WB with the indicated rmAbs (1 μg/ml), followed by anti-human IgG Ab (left panel) or anti-FLAG Ab (right panel). (D) ELISA screening of rmAbs for binding to CBX proteins. The heatmap represents the resulting OD values of the indicated rmAbs, with “+” indicating a saturated signal. (E) Reactivity of gddY mouse serum IgA with the CBX proteins. WB was performed as in (C) using sera of individual BALB/c or gddY mice as the primary Ab as indicated and anti-IgA Ab as the secondary Ab (left, top) or anti-FLAG Ab to confirm the presence of CBX proteins on the blot (left, bottom). Representative data are shown here. Venn diagram showing the percentage of gddY mice whose serum IgA reacted with CBX1, CBX3, or CBX5 (right panel) (n = 14). (F) ELISA determination of anti-CBX3 IgA Abs in sera from healthy controls (HC; n = 30) and patients with IgAN (IgAN; n = 70). ***: 0.0006 (Mann–Whitney U test). The dashed line shows 99% confidence interval (CI) of the control sera. One of two or three independent experiments with similar results is shown in each panel. Source data are available for this figure.

Journal: Life science alliance

Article Title: Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

doi: 10.26508/lsa.202402588

Figure Lengend Snippet: Figure 1. Identification of MC antigens reactive to IgA auto-Abs generated in gddY mice. (A) Detection of antigens for rmAbs derived from gddY mouse PBs in the lysates of gddY mouse primary MCs by WB. Split membranes, each containing proteins in one lane, were incubated with the indicated rmAbs (1 μg/ml), followed by an anti-human IgG Ab. (B) Immunoprecipitation of gddY mouse MC lysates with rmAb#66 or the control rmAb#NP. SDS–PAGE of the precipitates was developed using silver staining. The bands indicated by arrows were excised and analyzed using mass spectrometry. Those from the rmAb#66 precipitate included CBX1, CBX3, and CBX5, as indicated by arrows. (C) FLAG-tagged CBX1, CBX3, or CBX5 proteins transiently expressed in HEK293T cells were detected by WB with the indicated rmAbs (1 μg/ml), followed by anti-human IgG Ab (left panel) or anti-FLAG Ab (right panel). (D) ELISA screening of rmAbs for binding to CBX proteins. The heatmap represents the resulting OD values of the indicated rmAbs, with “+” indicating a saturated signal. (E) Reactivity of gddY mouse serum IgA with the CBX proteins. WB was performed as in (C) using sera of individual BALB/c or gddY mice as the primary Ab as indicated and anti-IgA Ab as the secondary Ab (left, top) or anti-FLAG Ab to confirm the presence of CBX proteins on the blot (left, bottom). Representative data are shown here. Venn diagram showing the percentage of gddY mice whose serum IgA reacted with CBX1, CBX3, or CBX5 (right panel) (n = 14). (F) ELISA determination of anti-CBX3 IgA Abs in sera from healthy controls (HC; n = 30) and patients with IgAN (IgAN; n = 70). ***: 0.0006 (Mann–Whitney U test). The dashed line shows 99% confidence interval (CI) of the control sera. One of two or three independent experiments with similar results is shown in each panel. Source data are available for this figure.

Article Snippet: For FCM, the following Abs were used: anti-mouse IgA-APC (clone 11- 44-2; Southern Biotech), anti-mouse CD73-biotin (clone TY/11.8; BioLegend), anti-mouse CD45-PerCP-Cy5.5 (clone 30-F11; BioLegend), anti-mouse CD31-PE (clone 390; BioLegend), anti-mouse TER119BV510 (clone TER-119; BioLegend), rabbit polyclonal anti-HP1γ (CBX3) Ab (2619; Cell Signaling Technology), rabbit monoclonal anti-HP1β (CBX1) Ab (8676; Cell Signaling Technology), rabbit IgG (for isotypematched control; 011-000-003; Jackson ImmunoResearch), antirabbit IgG-BV421 (clone Poly4064; BioLegend), and anti-human IgG Fcγ-Alexa Fluor 647 (709-606-098; Jackson ImmunoResearch).

Techniques: Generated, Derivative Assay, Incubation, Immunoprecipitation, Control, SDS Page, Silver Staining, Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Binding Assay, MANN-WHITNEY

Figure 2. CBX3 is an autoantigen expressed on the surfaces of MCs. (A, B) Representative histograms of FCM analysis of glomerular CD45– cells from BALB/c mice stained with Abs against CD45, CD73, and CD31 for gating on MCs (CD73+

Journal: Life science alliance

Article Title: Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

doi: 10.26508/lsa.202402588

Figure Lengend Snippet: Figure 2. CBX3 is an autoantigen expressed on the surfaces of MCs. (A, B) Representative histograms of FCM analysis of glomerular CD45– cells from BALB/c mice stained with Abs against CD45, CD73, and CD31 for gating on MCs (CD73+

Article Snippet: For FCM, the following Abs were used: anti-mouse IgA-APC (clone 11- 44-2; Southern Biotech), anti-mouse CD73-biotin (clone TY/11.8; BioLegend), anti-mouse CD45-PerCP-Cy5.5 (clone 30-F11; BioLegend), anti-mouse CD31-PE (clone 390; BioLegend), anti-mouse TER119BV510 (clone TER-119; BioLegend), rabbit polyclonal anti-HP1γ (CBX3) Ab (2619; Cell Signaling Technology), rabbit monoclonal anti-HP1β (CBX1) Ab (8676; Cell Signaling Technology), rabbit IgG (for isotypematched control; 011-000-003; Jackson ImmunoResearch), antirabbit IgG-BV421 (clone Poly4064; BioLegend), and anti-human IgG Fcγ-Alexa Fluor 647 (709-606-098; Jackson ImmunoResearch).

Techniques: Staining

Figure 5. The gddY auto-Abs reacted with commensal bacteria in the oral cavity of the gddY mice. (A, B) WB analysis of lysates of bacteria from feces (fecal), small intestine (SI), and oral cavity (oral) of gddY mice blotted with sera from gddY mice (A) or culture supernatant of leukocytes from gddY mouse kidneys (B) as the primary Abs and anti-IgA as the secondary Ab. The IgA concentration in the primary Abs was adjusted to 1 μg/ml by dilution. The bacteria were cultured anaerobically on brain heart infusion agar before lysis. (C, D, E, F) FCM analyses of cultured bacteria stained with various Ab solutions (containing 1 μg/ml IgA) and SYBR Green (for bacterial DNA). (C, D) Bacteria from the oral cavity (oral) or small intestine (C), or oral bacteria from BALB/c or gddY mice (D), were stained with sera from gddY mice. (E) Oral bacteria from gddY mice were stained with the culture supernatant of leukocytes from the indicated organs of gddY mice. (C, D, E) APC-conjugated anti-mouse IgA was used as secondary Ab. The secondary Ab alone did not stain cultured bacteria (data not shown). (F) Oral or fecal bacteria from gddY mice cultured under the indicated conditions were stained with rmAb#66 or rmAb#NP, followed by Alexa Fluor 647–anti-human IgG. (C, D, E) The frequencies of IgA-bound bacteria among whole bacteria stained with SYBR Green are plotted. Data are presented as means ± SD of biological replicates. (G) rmAb#66 (1 μg/ml) was incubated with the indicated concentrations of recombinant CBX3 or OVA in equal volumes for 20 min at RT, mixed with cultured oral bacteria from gddY mice, and incubated overnight on ice. The bacteria were stained with Alexa Fluor 647-anti-human IgG to detect bound rmAb#66, together with SYBR Green to label the bacteria, and analyzed by FCM. The frequencies of rmAb#66-bound bacteria among all bacteria were plotted. Data are presented as means ± SD of three technical replicates. *P < 0.05; ***P < 0.005; ****P < 0.001. P-values were calculated using unpaired t test. One of two independent experiments with similar results is shown in each panel. Source data are available for this figure.

Journal: Life science alliance

Article Title: Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

doi: 10.26508/lsa.202402588

Figure Lengend Snippet: Figure 5. The gddY auto-Abs reacted with commensal bacteria in the oral cavity of the gddY mice. (A, B) WB analysis of lysates of bacteria from feces (fecal), small intestine (SI), and oral cavity (oral) of gddY mice blotted with sera from gddY mice (A) or culture supernatant of leukocytes from gddY mouse kidneys (B) as the primary Abs and anti-IgA as the secondary Ab. The IgA concentration in the primary Abs was adjusted to 1 μg/ml by dilution. The bacteria were cultured anaerobically on brain heart infusion agar before lysis. (C, D, E, F) FCM analyses of cultured bacteria stained with various Ab solutions (containing 1 μg/ml IgA) and SYBR Green (for bacterial DNA). (C, D) Bacteria from the oral cavity (oral) or small intestine (C), or oral bacteria from BALB/c or gddY mice (D), were stained with sera from gddY mice. (E) Oral bacteria from gddY mice were stained with the culture supernatant of leukocytes from the indicated organs of gddY mice. (C, D, E) APC-conjugated anti-mouse IgA was used as secondary Ab. The secondary Ab alone did not stain cultured bacteria (data not shown). (F) Oral or fecal bacteria from gddY mice cultured under the indicated conditions were stained with rmAb#66 or rmAb#NP, followed by Alexa Fluor 647–anti-human IgG. (C, D, E) The frequencies of IgA-bound bacteria among whole bacteria stained with SYBR Green are plotted. Data are presented as means ± SD of biological replicates. (G) rmAb#66 (1 μg/ml) was incubated with the indicated concentrations of recombinant CBX3 or OVA in equal volumes for 20 min at RT, mixed with cultured oral bacteria from gddY mice, and incubated overnight on ice. The bacteria were stained with Alexa Fluor 647-anti-human IgG to detect bound rmAb#66, together with SYBR Green to label the bacteria, and analyzed by FCM. The frequencies of rmAb#66-bound bacteria among all bacteria were plotted. Data are presented as means ± SD of three technical replicates. *P < 0.05; ***P < 0.005; ****P < 0.001. P-values were calculated using unpaired t test. One of two independent experiments with similar results is shown in each panel. Source data are available for this figure.

Article Snippet: For FCM, the following Abs were used: anti-mouse IgA-APC (clone 11- 44-2; Southern Biotech), anti-mouse CD73-biotin (clone TY/11.8; BioLegend), anti-mouse CD45-PerCP-Cy5.5 (clone 30-F11; BioLegend), anti-mouse CD31-PE (clone 390; BioLegend), anti-mouse TER119BV510 (clone TER-119; BioLegend), rabbit polyclonal anti-HP1γ (CBX3) Ab (2619; Cell Signaling Technology), rabbit monoclonal anti-HP1β (CBX1) Ab (8676; Cell Signaling Technology), rabbit IgG (for isotypematched control; 011-000-003; Jackson ImmunoResearch), antirabbit IgG-BV421 (clone Poly4064; BioLegend), and anti-human IgG Fcγ-Alexa Fluor 647 (709-606-098; Jackson ImmunoResearch).

Techniques: Bacteria, Concentration Assay, Cell Culture, Lysis, Staining, SYBR Green Assay, Incubation, Recombinant

Figure 6. Identification of a bacterial strain that shares an rmAb#66 epitope with CBX3. (A) Isolated bacteria (C42) were stained with rmAb#66 or rmAb#NP (1 μg/ml), followed by Alexa Fluor 647–anti-human IgG together with SYBR Green, and analyzed by FCM. (B) rmAb#66 (1 μg/ml) was incubated with CBX3 or OVA and then with C42, which was stained with anti-human IgG and analyzed as described in Fig 5G. Data are presented as means ± SD of two technical replicates. (C) C42 bacteria (2 × 106 cells) were incubated with the indicated concentrations of PNGase F (Roche) for 4 h at 37°C, stained with rmAb#66 (1 μg/ml) overnight on ice, and labeled with SYBR Green. Binding of rmAb#66 to C42 was detected by FCM using Alexa Fluor 647-anti-human IgG (n = 2). (D) BALB/c mice were immunized s.c. with UV-killed C42 or E. faecalis, together with zymosan as an adjuvant, at 4 wk of age. WB analysis was performed as shown in Fig 3E (except that CBX1 and CBX5 samples were omitted) with sera from immunized mice at 8 wk of age as the primary Abs and anti-IgG Ab as the secondary Ab (left panel) or anti-FLAG Ab (right panel). (E) Representative IFM images of kidney sections from the mice used in (D). Kidney sections were stained with an anti-IgA Ab (red). Dashed circles indicate areas of glomeruli and white lines indicate scale bars (100 μm). (F) Frequency of bacteria recognized by rmAb#66 among all bacteria (SYBR Green+) in the oral cavity of gddY mice (n = 6). Bacteria from gddY mice fed a normal diet (control) or an ED (ED) were cultured anaerobically on brain heart infusion agar, stained with rmAb#66 (1 μg/ml) and Alexa Fluor 647–anti-human IgG, together with SYBR Green, and analyzed by FCM. (G) Frequency of each bacterial strain among the total bacteria in the oral cavities of gddY mice fed a normal diet (control) or an ED, as determined by 16S rRNA bulk sequencing (n = 6). An underscore after each genus name indicates that the species was not classified. Data are presented as means ± SD of biological replicates. *P < 0.05; ***P < 0.005; ****P < 0.001. P-values were calculated using the unpaired t test. One of two independent experiments with similar results is shown in each panel. Source data are available for this figure.

Journal: Life science alliance

Article Title: Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

doi: 10.26508/lsa.202402588

Figure Lengend Snippet: Figure 6. Identification of a bacterial strain that shares an rmAb#66 epitope with CBX3. (A) Isolated bacteria (C42) were stained with rmAb#66 or rmAb#NP (1 μg/ml), followed by Alexa Fluor 647–anti-human IgG together with SYBR Green, and analyzed by FCM. (B) rmAb#66 (1 μg/ml) was incubated with CBX3 or OVA and then with C42, which was stained with anti-human IgG and analyzed as described in Fig 5G. Data are presented as means ± SD of two technical replicates. (C) C42 bacteria (2 × 106 cells) were incubated with the indicated concentrations of PNGase F (Roche) for 4 h at 37°C, stained with rmAb#66 (1 μg/ml) overnight on ice, and labeled with SYBR Green. Binding of rmAb#66 to C42 was detected by FCM using Alexa Fluor 647-anti-human IgG (n = 2). (D) BALB/c mice were immunized s.c. with UV-killed C42 or E. faecalis, together with zymosan as an adjuvant, at 4 wk of age. WB analysis was performed as shown in Fig 3E (except that CBX1 and CBX5 samples were omitted) with sera from immunized mice at 8 wk of age as the primary Abs and anti-IgG Ab as the secondary Ab (left panel) or anti-FLAG Ab (right panel). (E) Representative IFM images of kidney sections from the mice used in (D). Kidney sections were stained with an anti-IgA Ab (red). Dashed circles indicate areas of glomeruli and white lines indicate scale bars (100 μm). (F) Frequency of bacteria recognized by rmAb#66 among all bacteria (SYBR Green+) in the oral cavity of gddY mice (n = 6). Bacteria from gddY mice fed a normal diet (control) or an ED (ED) were cultured anaerobically on brain heart infusion agar, stained with rmAb#66 (1 μg/ml) and Alexa Fluor 647–anti-human IgG, together with SYBR Green, and analyzed by FCM. (G) Frequency of each bacterial strain among the total bacteria in the oral cavities of gddY mice fed a normal diet (control) or an ED, as determined by 16S rRNA bulk sequencing (n = 6). An underscore after each genus name indicates that the species was not classified. Data are presented as means ± SD of biological replicates. *P < 0.05; ***P < 0.005; ****P < 0.001. P-values were calculated using the unpaired t test. One of two independent experiments with similar results is shown in each panel. Source data are available for this figure.

Article Snippet: For FCM, the following Abs were used: anti-mouse IgA-APC (clone 11- 44-2; Southern Biotech), anti-mouse CD73-biotin (clone TY/11.8; BioLegend), anti-mouse CD45-PerCP-Cy5.5 (clone 30-F11; BioLegend), anti-mouse CD31-PE (clone 390; BioLegend), anti-mouse TER119BV510 (clone TER-119; BioLegend), rabbit polyclonal anti-HP1γ (CBX3) Ab (2619; Cell Signaling Technology), rabbit monoclonal anti-HP1β (CBX1) Ab (8676; Cell Signaling Technology), rabbit IgG (for isotypematched control; 011-000-003; Jackson ImmunoResearch), antirabbit IgG-BV421 (clone Poly4064; BioLegend), and anti-human IgG Fcγ-Alexa Fluor 647 (709-606-098; Jackson ImmunoResearch).

Techniques: Isolation, Bacteria, Staining, SYBR Green Assay, Incubation, Labeling, Binding Assay, Adjuvant, Control, Cell Culture, Sequencing

Figure 7. Determination of rmAb#66 epitope on CBX3. (A) Schematic representation of CBX3 proteins: 1/2 CBX3 (AA1-92) and 2/3 CBX3 (AA63-184). (B, C) FLAG-tagged 1/2 or 2/3 CBX3 proteins, transiently expressed in HEK293T cells, were detected by WB with rmAb#66 or rmAb#NP (1 μg/ml) followed by anti-human IgG Ab ((B), left panel). The same membrane was reblotted with sera of two gddY mice followed by anti-mouse IgA Ab ((C), left panel). Protein loading was confirmed using an anti-FLAG Ab ((B, C), right panels; the same blot is shown). (D) Amino acid sequence alignment of CBX proteins (AA1-62) (top). WB analysis of FLAG-tagged WT or the indicated mutated CBX3 proteins transiently expressed in HEK293T cells, performed as in (B) (bottom). (E) Reactivity to CBX3 (WT) or CBX3 mutant 54FA (54FA) proteins of the indicated rmAbs at the indicated concentrations was evaluated by ELISA and presented as OD450 values. One of two independent experiments with similar results is shown in each panel. Source data are available for this figure.

Journal: Life science alliance

Article Title: Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

doi: 10.26508/lsa.202402588

Figure Lengend Snippet: Figure 7. Determination of rmAb#66 epitope on CBX3. (A) Schematic representation of CBX3 proteins: 1/2 CBX3 (AA1-92) and 2/3 CBX3 (AA63-184). (B, C) FLAG-tagged 1/2 or 2/3 CBX3 proteins, transiently expressed in HEK293T cells, were detected by WB with rmAb#66 or rmAb#NP (1 μg/ml) followed by anti-human IgG Ab ((B), left panel). The same membrane was reblotted with sera of two gddY mice followed by anti-mouse IgA Ab ((C), left panel). Protein loading was confirmed using an anti-FLAG Ab ((B, C), right panels; the same blot is shown). (D) Amino acid sequence alignment of CBX proteins (AA1-62) (top). WB analysis of FLAG-tagged WT or the indicated mutated CBX3 proteins transiently expressed in HEK293T cells, performed as in (B) (bottom). (E) Reactivity to CBX3 (WT) or CBX3 mutant 54FA (54FA) proteins of the indicated rmAbs at the indicated concentrations was evaluated by ELISA and presented as OD450 values. One of two independent experiments with similar results is shown in each panel. Source data are available for this figure.

Article Snippet: For FCM, the following Abs were used: anti-mouse IgA-APC (clone 11- 44-2; Southern Biotech), anti-mouse CD73-biotin (clone TY/11.8; BioLegend), anti-mouse CD45-PerCP-Cy5.5 (clone 30-F11; BioLegend), anti-mouse CD31-PE (clone 390; BioLegend), anti-mouse TER119BV510 (clone TER-119; BioLegend), rabbit polyclonal anti-HP1γ (CBX3) Ab (2619; Cell Signaling Technology), rabbit monoclonal anti-HP1β (CBX1) Ab (8676; Cell Signaling Technology), rabbit IgG (for isotypematched control; 011-000-003; Jackson ImmunoResearch), antirabbit IgG-BV421 (clone Poly4064; BioLegend), and anti-human IgG Fcγ-Alexa Fluor 647 (709-606-098; Jackson ImmunoResearch).

Techniques: Membrane, Sequencing, Mutagenesis, Enzyme-linked Immunosorbent Assay

( A ) FLAG-tagged CBX3 was immunoprecipitated from 293T cells and subjected to MS analysis to identify CBX3 interacting proteins. ( B ) The list of top hits proteins from CBX3 MS. ( C ) Western blot (WB) analysis of whole-cell lysates (WCL) and anti-CBX3 immunoprecipitations (IPs) derived from PC-3 cells. ( D ) WB analysis of WCL and anti-RPA2 IPs derived from PC-3 cells. ( E ) Colocalization of CBX3 with RPA2 foci after noted drug treatment or transfected with indicated small interfering RNA (siRNA) in U2OS cells. Scale bar, 10 μm. ( F ) Quantitation of average CBX3 foci number in each cell. Data were presented as means ± SD of more than 200 cells from three biological replicates. n.s., not significant. Two-tailed unpaired Student’s t test, *** P < 0.001. ( G ) Replication fork-bound proteins were isolated by the iPOND assay with control and RPA2 knockdown U2OS cells. ( H ) Schematic diagram depicting a set of GST-RPA2 recombinant protein constructs. ( I ) WB analysis of Flag-CBX3 proteins in U2OS cells pulled down by GST or GST-RPA2 recombinant proteins. ( J ) Schematic diagram depicting a set of GST-CBX3 recombinant protein constructs. ( K ) WB of His-RPA2 proteins in U2OS cells pulled down by GST or GST-CBX3 recombinant proteins. ( L and M ) U2OS cells transfected with Flag-CBX3 proteins were subjected to immunofluorescence staining (IFC) after HU treatment. Representative images were shown in (L) and quantitative data are shown in (M). Scale bar, 10 μm. Data were shown as the mean ± SD from three biological replicates, two-tailed unpaired Student’s t test, *** P < 0.001. IgG, immunoglobulin.

Journal: Science Advances

Article Title: CK2-dependent degradation of CBX3 dictates replication fork stalling and PARP inhibitor sensitivity

doi: 10.1126/sciadv.adk8908

Figure Lengend Snippet: ( A ) FLAG-tagged CBX3 was immunoprecipitated from 293T cells and subjected to MS analysis to identify CBX3 interacting proteins. ( B ) The list of top hits proteins from CBX3 MS. ( C ) Western blot (WB) analysis of whole-cell lysates (WCL) and anti-CBX3 immunoprecipitations (IPs) derived from PC-3 cells. ( D ) WB analysis of WCL and anti-RPA2 IPs derived from PC-3 cells. ( E ) Colocalization of CBX3 with RPA2 foci after noted drug treatment or transfected with indicated small interfering RNA (siRNA) in U2OS cells. Scale bar, 10 μm. ( F ) Quantitation of average CBX3 foci number in each cell. Data were presented as means ± SD of more than 200 cells from three biological replicates. n.s., not significant. Two-tailed unpaired Student’s t test, *** P < 0.001. ( G ) Replication fork-bound proteins were isolated by the iPOND assay with control and RPA2 knockdown U2OS cells. ( H ) Schematic diagram depicting a set of GST-RPA2 recombinant protein constructs. ( I ) WB analysis of Flag-CBX3 proteins in U2OS cells pulled down by GST or GST-RPA2 recombinant proteins. ( J ) Schematic diagram depicting a set of GST-CBX3 recombinant protein constructs. ( K ) WB of His-RPA2 proteins in U2OS cells pulled down by GST or GST-CBX3 recombinant proteins. ( L and M ) U2OS cells transfected with Flag-CBX3 proteins were subjected to immunofluorescence staining (IFC) after HU treatment. Representative images were shown in (L) and quantitative data are shown in (M). Scale bar, 10 μm. Data were shown as the mean ± SD from three biological replicates, two-tailed unpaired Student’s t test, *** P < 0.001. IgG, immunoglobulin.

Article Snippet: Primary antibodies used were CBX3 (1:1000; Santa Cruz Biotechnology, catalog no. sc-398562), CBX1 (1:1000; Proteintech Group catalog no. 10241-2-AP), CBX5 (1:1000; Proteintech Group catalog no. 11831-1-AP), RPA2 (1:500; Cell Signaling Technology, catalog no. 35869), RPA1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-271564), RPA3 (1:1000; Cell Signaling Technology, catalog no. 42206), HA.11 (1:1000; Covance, catalog no. MMS-101R), RFWD3 (1:1000; Abcam, catalog no. ab222149), UNG2 (1:500; Santa Cruz Biotechnology, catalog no. sc-390255), SMARCAL1 (1:500; Santa Cruz Biotechnology, catalog no. sc-376377), Myc (1:1000; Santa Cruz Biotechnology, catalog no. sc-40), Flag (1:1000; Sigma-Aldrich, catalog no. F-3165), extracellular signal–regulated kinase 2 (1:1000; Santa Cruz Biotechnology, catalog no. sc-1647), phospho Histone H2A.X (S139) (1:1000; Cell Signaling Technology, catalog no. 9718), phospho Histone H2A.X (S139) (1:1000; Cell Signaling Technology, catalog no. 80312S), phospho-RPA2 (S4/S8) (1:1000; Abcam, catalog no. ab87277), phospho-RPA2 (T21) (1:1000; Abcam, catalog no. ab61065), CHK1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-8408), phospho CHK1 (S345) (1:1000; Cell Signaling Technology, catalog no. 2348), ubiquitin (1:1000; Santa Cruz Biotechnology, catalog no. sc-8017), glyceraldehyde-3-phosphate dehydrogenase (1:1000; Santa Cruz Biotechnology, catalog no. sc-47724), CDH1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-56312), CDC20 (1:1000; Santa Cruz Biotechnology, catalog no. sc-13162), and His (1:1000; Santa Cruz Biotechnology, catalog no. sc-53073).

Techniques: Immunoprecipitation, Western Blot, Derivative Assay, Transfection, Small Interfering RNA, Quantitation Assay, Two Tailed Test, Isolation, Control, Knockdown, Recombinant, Construct, Immunofluorescence, Staining

( A and B ) U2OS cells infected with lentivirus expressing control or sh CBX3 were subjected to IFC after HU treatment. Representative images were shown in (A) and quantitative data are shown in (B). Scale bar, 10 μm. Data were shown as the mean ± SD from three biological replicates, two-tailed unpaired Student's t test, *** P < 0.001. ( C ) Replication fork-bound proteins were isolated by the iPOND assay with control and CBX3 knockdown U2OS cells. ( D ) WB analysis of WCL and anti-His IPs derived from PC-3 cells transfected with indicated plasmids followed by HU treatment. ( E ) WB analysis of WCL and anti-RPA2 IPs derived from PC-3 control and CBX3 knockdown cells transfected with indicated plasmids followed by HU treatment. ( F ) WB analysis of WCL and anti-His IPs derived from PC-3 cells transfected with indicated plasmids followed by HU treatment. Both poly-ubiquitinated and primary-ubiquitinated RPA2 (asterisk) were observed. ( G ) WB analysis of WCL and anti-RPA2 IPs derived from PC-3 control and CBX3 knockdown cells transfected with indicated plasmids followed by HU treatment. Both poly-ubiquitinated and primary-ubiquitinated RPA2 (asterisk) were observed. ( H and I ) U2OS control or CBX3 knockdown cells were reconstituted with WT or V22M mutant CBX3 and subjected to IFC after HU treatment. Representative images were shown in (H) and quantitative data were shown in (I). Scale bar, 10 μm. Data were shown as the means ± SD from three biological replicates, two-tailed unpaired Student's t test, *** P < 0.001. ( J ) WB analysis of WCL and chromatin binding proteins derived from PC-3 control and CBX3 knockdown cells transfected with indicated plasmids followed by HU treatment.

Journal: Science Advances

Article Title: CK2-dependent degradation of CBX3 dictates replication fork stalling and PARP inhibitor sensitivity

doi: 10.1126/sciadv.adk8908

Figure Lengend Snippet: ( A and B ) U2OS cells infected with lentivirus expressing control or sh CBX3 were subjected to IFC after HU treatment. Representative images were shown in (A) and quantitative data are shown in (B). Scale bar, 10 μm. Data were shown as the mean ± SD from three biological replicates, two-tailed unpaired Student's t test, *** P < 0.001. ( C ) Replication fork-bound proteins were isolated by the iPOND assay with control and CBX3 knockdown U2OS cells. ( D ) WB analysis of WCL and anti-His IPs derived from PC-3 cells transfected with indicated plasmids followed by HU treatment. ( E ) WB analysis of WCL and anti-RPA2 IPs derived from PC-3 control and CBX3 knockdown cells transfected with indicated plasmids followed by HU treatment. ( F ) WB analysis of WCL and anti-His IPs derived from PC-3 cells transfected with indicated plasmids followed by HU treatment. Both poly-ubiquitinated and primary-ubiquitinated RPA2 (asterisk) were observed. ( G ) WB analysis of WCL and anti-RPA2 IPs derived from PC-3 control and CBX3 knockdown cells transfected with indicated plasmids followed by HU treatment. Both poly-ubiquitinated and primary-ubiquitinated RPA2 (asterisk) were observed. ( H and I ) U2OS control or CBX3 knockdown cells were reconstituted with WT or V22M mutant CBX3 and subjected to IFC after HU treatment. Representative images were shown in (H) and quantitative data were shown in (I). Scale bar, 10 μm. Data were shown as the means ± SD from three biological replicates, two-tailed unpaired Student's t test, *** P < 0.001. ( J ) WB analysis of WCL and chromatin binding proteins derived from PC-3 control and CBX3 knockdown cells transfected with indicated plasmids followed by HU treatment.

Article Snippet: Primary antibodies used were CBX3 (1:1000; Santa Cruz Biotechnology, catalog no. sc-398562), CBX1 (1:1000; Proteintech Group catalog no. 10241-2-AP), CBX5 (1:1000; Proteintech Group catalog no. 11831-1-AP), RPA2 (1:500; Cell Signaling Technology, catalog no. 35869), RPA1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-271564), RPA3 (1:1000; Cell Signaling Technology, catalog no. 42206), HA.11 (1:1000; Covance, catalog no. MMS-101R), RFWD3 (1:1000; Abcam, catalog no. ab222149), UNG2 (1:500; Santa Cruz Biotechnology, catalog no. sc-390255), SMARCAL1 (1:500; Santa Cruz Biotechnology, catalog no. sc-376377), Myc (1:1000; Santa Cruz Biotechnology, catalog no. sc-40), Flag (1:1000; Sigma-Aldrich, catalog no. F-3165), extracellular signal–regulated kinase 2 (1:1000; Santa Cruz Biotechnology, catalog no. sc-1647), phospho Histone H2A.X (S139) (1:1000; Cell Signaling Technology, catalog no. 9718), phospho Histone H2A.X (S139) (1:1000; Cell Signaling Technology, catalog no. 80312S), phospho-RPA2 (S4/S8) (1:1000; Abcam, catalog no. ab87277), phospho-RPA2 (T21) (1:1000; Abcam, catalog no. ab61065), CHK1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-8408), phospho CHK1 (S345) (1:1000; Cell Signaling Technology, catalog no. 2348), ubiquitin (1:1000; Santa Cruz Biotechnology, catalog no. sc-8017), glyceraldehyde-3-phosphate dehydrogenase (1:1000; Santa Cruz Biotechnology, catalog no. sc-47724), CDH1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-56312), CDC20 (1:1000; Santa Cruz Biotechnology, catalog no. sc-13162), and His (1:1000; Santa Cruz Biotechnology, catalog no. sc-53073).

Techniques: Infection, Expressing, Control, Two Tailed Test, Isolation, Knockdown, Derivative Assay, Transfection, Mutagenesis, Binding Assay

( A ) PC-3 cells were synchronized by thymidine and nocodazole double blockade. Cells were released and harvested at the indicated time points for WB analysis. ( B ) WB analysis of WCL and anti-CBX3 IPs derived from PC-3 cells. ( C ) WB analysis of WCL and anti-CDH1 IPs derived from PC-3 cells. ( D ) WB analysis of WCL derived from PC-3 cells transfected with indicated plasmids. ( E ) WB analysis of WCL derived from PC-3 control and CDH1 knockdown cells. ( F and G ) PC-3 control or CDH1 knockdown cells were treated with CHX (50 μg/μl) for WB (F). Protein bands were quantified as in (G). ( H ) WB analysis of WCL and anti-CBX3 IPs derived from PC-3 cells transfected with indicated plasmids followed by MG132 treatment. Both poly-ubiquitinated and primary-ubiquitinated CBX3 (asterisk) were observed. ( I ) WB analysis of WCL and anti-CBX3 IPs derived from PC-3 control and CDH1 knockdown cells transfected with indicated plasmids followed by MG132 treatment. Both poly-ubiquitinated and primary-ubiquitinated CBX3 (asterisk) were observed. ( J ) Diagram showing predicted D-box from the online APC/C degron repository in CBX3. ( K ) WB analysis of WCL and anti-Flag IPs derived from PC-3 cells transfected with indicated plasmids. ( L and M ) PC-3 cells transfected with indicated plasmids were treated with CHX (50 μg/μl) for WB (L). Protein bands were quantified as in (M).

Journal: Science Advances

Article Title: CK2-dependent degradation of CBX3 dictates replication fork stalling and PARP inhibitor sensitivity

doi: 10.1126/sciadv.adk8908

Figure Lengend Snippet: ( A ) PC-3 cells were synchronized by thymidine and nocodazole double blockade. Cells were released and harvested at the indicated time points for WB analysis. ( B ) WB analysis of WCL and anti-CBX3 IPs derived from PC-3 cells. ( C ) WB analysis of WCL and anti-CDH1 IPs derived from PC-3 cells. ( D ) WB analysis of WCL derived from PC-3 cells transfected with indicated plasmids. ( E ) WB analysis of WCL derived from PC-3 control and CDH1 knockdown cells. ( F and G ) PC-3 control or CDH1 knockdown cells were treated with CHX (50 μg/μl) for WB (F). Protein bands were quantified as in (G). ( H ) WB analysis of WCL and anti-CBX3 IPs derived from PC-3 cells transfected with indicated plasmids followed by MG132 treatment. Both poly-ubiquitinated and primary-ubiquitinated CBX3 (asterisk) were observed. ( I ) WB analysis of WCL and anti-CBX3 IPs derived from PC-3 control and CDH1 knockdown cells transfected with indicated plasmids followed by MG132 treatment. Both poly-ubiquitinated and primary-ubiquitinated CBX3 (asterisk) were observed. ( J ) Diagram showing predicted D-box from the online APC/C degron repository in CBX3. ( K ) WB analysis of WCL and anti-Flag IPs derived from PC-3 cells transfected with indicated plasmids. ( L and M ) PC-3 cells transfected with indicated plasmids were treated with CHX (50 μg/μl) for WB (L). Protein bands were quantified as in (M).

Article Snippet: Primary antibodies used were CBX3 (1:1000; Santa Cruz Biotechnology, catalog no. sc-398562), CBX1 (1:1000; Proteintech Group catalog no. 10241-2-AP), CBX5 (1:1000; Proteintech Group catalog no. 11831-1-AP), RPA2 (1:500; Cell Signaling Technology, catalog no. 35869), RPA1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-271564), RPA3 (1:1000; Cell Signaling Technology, catalog no. 42206), HA.11 (1:1000; Covance, catalog no. MMS-101R), RFWD3 (1:1000; Abcam, catalog no. ab222149), UNG2 (1:500; Santa Cruz Biotechnology, catalog no. sc-390255), SMARCAL1 (1:500; Santa Cruz Biotechnology, catalog no. sc-376377), Myc (1:1000; Santa Cruz Biotechnology, catalog no. sc-40), Flag (1:1000; Sigma-Aldrich, catalog no. F-3165), extracellular signal–regulated kinase 2 (1:1000; Santa Cruz Biotechnology, catalog no. sc-1647), phospho Histone H2A.X (S139) (1:1000; Cell Signaling Technology, catalog no. 9718), phospho Histone H2A.X (S139) (1:1000; Cell Signaling Technology, catalog no. 80312S), phospho-RPA2 (S4/S8) (1:1000; Abcam, catalog no. ab87277), phospho-RPA2 (T21) (1:1000; Abcam, catalog no. ab61065), CHK1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-8408), phospho CHK1 (S345) (1:1000; Cell Signaling Technology, catalog no. 2348), ubiquitin (1:1000; Santa Cruz Biotechnology, catalog no. sc-8017), glyceraldehyde-3-phosphate dehydrogenase (1:1000; Santa Cruz Biotechnology, catalog no. sc-47724), CDH1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-56312), CDC20 (1:1000; Santa Cruz Biotechnology, catalog no. sc-13162), and His (1:1000; Santa Cruz Biotechnology, catalog no. sc-53073).

Techniques: Derivative Assay, Transfection, Control, Knockdown

( A ) WB analysis of Flag-CBX3 proteins in PC-3 cells pulled down by GST or GST-CDH1 recombinant proteins. ( B ) Diagram showing phosphorylation motifs from the D-box in CBX3. ( C ) WB analysis of WCL derived from PC-3 control and CK2 knockdown cells. ( D and E ) PC-3 control or CK2 knockdown cells were treated with CHX (50 μg/μl) for WB (D). Protein bands were quantified as in (E). ( F ) MS analysis reveals phosphorylation of CBX3 serine-95. ( G ) WB analysis of WCL and anti-Flag IPs derived from PC-3 cells transfected with indicated plasmids. ( H ) WB analysis of WCL and anti-Flag IPs derived from PC-3 cells transfected with indicated plasmids. ( I and J ) PC-3 cells transfected with indicated plasmids were treated with CHX (50 μg/μl) for WB (I). Protein bands were quantified as in (J). ( K to M ) U2OS control or CBX3 knockdown cells were reconstituted with WT or V22M mutant CBX3 and subjected to IFC after HU treatment. Representative images were shown in (K). Scale bar, 10 μm. Quantitative data of RFWD3 foci were shown in (L) and RPA2 foci were shown in (M). Data were shown as the mean ± SD from three biological replicates, two-tailed unpaired Student’s t test, *** P < 0.001.

Journal: Science Advances

Article Title: CK2-dependent degradation of CBX3 dictates replication fork stalling and PARP inhibitor sensitivity

doi: 10.1126/sciadv.adk8908

Figure Lengend Snippet: ( A ) WB analysis of Flag-CBX3 proteins in PC-3 cells pulled down by GST or GST-CDH1 recombinant proteins. ( B ) Diagram showing phosphorylation motifs from the D-box in CBX3. ( C ) WB analysis of WCL derived from PC-3 control and CK2 knockdown cells. ( D and E ) PC-3 control or CK2 knockdown cells were treated with CHX (50 μg/μl) for WB (D). Protein bands were quantified as in (E). ( F ) MS analysis reveals phosphorylation of CBX3 serine-95. ( G ) WB analysis of WCL and anti-Flag IPs derived from PC-3 cells transfected with indicated plasmids. ( H ) WB analysis of WCL and anti-Flag IPs derived from PC-3 cells transfected with indicated plasmids. ( I and J ) PC-3 cells transfected with indicated plasmids were treated with CHX (50 μg/μl) for WB (I). Protein bands were quantified as in (J). ( K to M ) U2OS control or CBX3 knockdown cells were reconstituted with WT or V22M mutant CBX3 and subjected to IFC after HU treatment. Representative images were shown in (K). Scale bar, 10 μm. Quantitative data of RFWD3 foci were shown in (L) and RPA2 foci were shown in (M). Data were shown as the mean ± SD from three biological replicates, two-tailed unpaired Student’s t test, *** P < 0.001.

Article Snippet: Primary antibodies used were CBX3 (1:1000; Santa Cruz Biotechnology, catalog no. sc-398562), CBX1 (1:1000; Proteintech Group catalog no. 10241-2-AP), CBX5 (1:1000; Proteintech Group catalog no. 11831-1-AP), RPA2 (1:500; Cell Signaling Technology, catalog no. 35869), RPA1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-271564), RPA3 (1:1000; Cell Signaling Technology, catalog no. 42206), HA.11 (1:1000; Covance, catalog no. MMS-101R), RFWD3 (1:1000; Abcam, catalog no. ab222149), UNG2 (1:500; Santa Cruz Biotechnology, catalog no. sc-390255), SMARCAL1 (1:500; Santa Cruz Biotechnology, catalog no. sc-376377), Myc (1:1000; Santa Cruz Biotechnology, catalog no. sc-40), Flag (1:1000; Sigma-Aldrich, catalog no. F-3165), extracellular signal–regulated kinase 2 (1:1000; Santa Cruz Biotechnology, catalog no. sc-1647), phospho Histone H2A.X (S139) (1:1000; Cell Signaling Technology, catalog no. 9718), phospho Histone H2A.X (S139) (1:1000; Cell Signaling Technology, catalog no. 80312S), phospho-RPA2 (S4/S8) (1:1000; Abcam, catalog no. ab87277), phospho-RPA2 (T21) (1:1000; Abcam, catalog no. ab61065), CHK1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-8408), phospho CHK1 (S345) (1:1000; Cell Signaling Technology, catalog no. 2348), ubiquitin (1:1000; Santa Cruz Biotechnology, catalog no. sc-8017), glyceraldehyde-3-phosphate dehydrogenase (1:1000; Santa Cruz Biotechnology, catalog no. sc-47724), CDH1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-56312), CDC20 (1:1000; Santa Cruz Biotechnology, catalog no. sc-13162), and His (1:1000; Santa Cruz Biotechnology, catalog no. sc-53073).

Techniques: Recombinant, Phospho-proteomics, Derivative Assay, Control, Knockdown, Transfection, Mutagenesis, Two Tailed Test

( A ) PC-3 and U2OS cells infected with lentivirus expressing EV or CBX3 were treated with or without HU followed by WB. ( B ) PC-3 and U2OS cells were treated with indicated agents followed by WB. ( C ) Representative images of normal, delayed or failed DNA replication restart detected by DNA fiber assay in PC-3 and U2OS cells. Scale bar, 20 μm. ( D and E ) Analysis of IdU tract length compared to CldU tract length from DNA fiber assay in PC-3 and U2OS EV and CBX3-OE (D) or CK2 inhibitor-treated (E) cells. Two-tailed unpaired Student’s t test, *** P < 0.001. ( F and G ) Stalled forks from DNA fiber assay in PC-3 and U2OS EV and CBX3-OE (F) or CK2 inhibitor-treated (G) cells. Two-tailed unpaired Student’s t test, *** P < 0.001. ( H to J ) Representative images of IHC staining (H) of indicated antibodies on prostate cancer patient specimens ( n = 40). Scale bar in 40 X fields: 50 μm. Quantification of γ-H2AX IHC score was shown in (I and J). ( K and L ) PC-3 cells infected with lentivirus expressing EV or CBX3 were subjected to γ-H2AX IFC (K). The average γ-H2AX foci number in each cell was quantified (L). Scale bar, 50 μm. n.s., not significant. Two-tailed unpaired Student’s t test, *** P < 0.001. ( M ) Analysis of HRD score, LST, LOH, and TAI in prostate cancer samples (see STAR Methods section). ( N ) Analysis of the correlation between tumor mutation burden and CBX3 mRNA levels from TCGA prostate cancer dataset. ( O and P ) PC-3 cells infected with lentivirus expressing EV or CBX3 were treated with HU (2 nM) for 5 hours followed by karyotyping. Representative images were shown in (O) and quantitative data were shown in (P). Scale bar, 10 μm. Two-tailed unpaired Student’s t test, *** P < 0.001.

Journal: Science Advances

Article Title: CK2-dependent degradation of CBX3 dictates replication fork stalling and PARP inhibitor sensitivity

doi: 10.1126/sciadv.adk8908

Figure Lengend Snippet: ( A ) PC-3 and U2OS cells infected with lentivirus expressing EV or CBX3 were treated with or without HU followed by WB. ( B ) PC-3 and U2OS cells were treated with indicated agents followed by WB. ( C ) Representative images of normal, delayed or failed DNA replication restart detected by DNA fiber assay in PC-3 and U2OS cells. Scale bar, 20 μm. ( D and E ) Analysis of IdU tract length compared to CldU tract length from DNA fiber assay in PC-3 and U2OS EV and CBX3-OE (D) or CK2 inhibitor-treated (E) cells. Two-tailed unpaired Student’s t test, *** P < 0.001. ( F and G ) Stalled forks from DNA fiber assay in PC-3 and U2OS EV and CBX3-OE (F) or CK2 inhibitor-treated (G) cells. Two-tailed unpaired Student’s t test, *** P < 0.001. ( H to J ) Representative images of IHC staining (H) of indicated antibodies on prostate cancer patient specimens ( n = 40). Scale bar in 40 X fields: 50 μm. Quantification of γ-H2AX IHC score was shown in (I and J). ( K and L ) PC-3 cells infected with lentivirus expressing EV or CBX3 were subjected to γ-H2AX IFC (K). The average γ-H2AX foci number in each cell was quantified (L). Scale bar, 50 μm. n.s., not significant. Two-tailed unpaired Student’s t test, *** P < 0.001. ( M ) Analysis of HRD score, LST, LOH, and TAI in prostate cancer samples (see STAR Methods section). ( N ) Analysis of the correlation between tumor mutation burden and CBX3 mRNA levels from TCGA prostate cancer dataset. ( O and P ) PC-3 cells infected with lentivirus expressing EV or CBX3 were treated with HU (2 nM) for 5 hours followed by karyotyping. Representative images were shown in (O) and quantitative data were shown in (P). Scale bar, 10 μm. Two-tailed unpaired Student’s t test, *** P < 0.001.

Article Snippet: Primary antibodies used were CBX3 (1:1000; Santa Cruz Biotechnology, catalog no. sc-398562), CBX1 (1:1000; Proteintech Group catalog no. 10241-2-AP), CBX5 (1:1000; Proteintech Group catalog no. 11831-1-AP), RPA2 (1:500; Cell Signaling Technology, catalog no. 35869), RPA1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-271564), RPA3 (1:1000; Cell Signaling Technology, catalog no. 42206), HA.11 (1:1000; Covance, catalog no. MMS-101R), RFWD3 (1:1000; Abcam, catalog no. ab222149), UNG2 (1:500; Santa Cruz Biotechnology, catalog no. sc-390255), SMARCAL1 (1:500; Santa Cruz Biotechnology, catalog no. sc-376377), Myc (1:1000; Santa Cruz Biotechnology, catalog no. sc-40), Flag (1:1000; Sigma-Aldrich, catalog no. F-3165), extracellular signal–regulated kinase 2 (1:1000; Santa Cruz Biotechnology, catalog no. sc-1647), phospho Histone H2A.X (S139) (1:1000; Cell Signaling Technology, catalog no. 9718), phospho Histone H2A.X (S139) (1:1000; Cell Signaling Technology, catalog no. 80312S), phospho-RPA2 (S4/S8) (1:1000; Abcam, catalog no. ab87277), phospho-RPA2 (T21) (1:1000; Abcam, catalog no. ab61065), CHK1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-8408), phospho CHK1 (S345) (1:1000; Cell Signaling Technology, catalog no. 2348), ubiquitin (1:1000; Santa Cruz Biotechnology, catalog no. sc-8017), glyceraldehyde-3-phosphate dehydrogenase (1:1000; Santa Cruz Biotechnology, catalog no. sc-47724), CDH1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-56312), CDC20 (1:1000; Santa Cruz Biotechnology, catalog no. sc-13162), and His (1:1000; Santa Cruz Biotechnology, catalog no. sc-53073).

Techniques: Infection, Expressing, Two Tailed Test, Immunohistochemistry, Mutagenesis

( A ) Dose-response survival curves of EV or CBX3 expressing cells exposed to increasing concentrations of olaparib in PC-3 cells. ( B and C ) Colony formation assays from PC-3 cell lines infected with lentivirus expressing EV or CBX3. Representative colonies are shown in (B) with quantification data shown in (C). Scale bar, 5 mm. ( D to F ) PC-3 cells infected with lentivirus expressing EV or CBX3 were injected subcutaneously into the right flank of SCID mice and treated with vehicle or olaparib (50 mg/kg). Tumors were harvested, photographed and shown in (D). Tumor volume was measured in (E). Tumor weight was measured in (F). ( G ) WB analysis of WCL derived from PC-3 and U2OS cells treated with indicated inhibitors. ( H ) Dose-response survival curves of control or CK2 inhibitor treated cells exposed to increasing concentrations of olaparib in PC-3 cells. ( I and J ) Colony formation assays were performed in PC-3 cell lines treated with indicated inhibitor. Representative colonies were shown in (I) with quantification data shown in (J). Scale bar, 5 mm. ( K to M ) PC-3 cells were injected subcutaneously into the right flank of SCID mice and treated with vehicle, CK2 inhibitor (75 mg/kg), olaparib (50 mg/kg), or combination. Tumors in each group at day 49 were harvested, and photographed shown in (K). Tumor volume (L). Tumor weight (M). ( N and O ) Representative images of IHC staining of γ-H2AX on PC-3 tumor tissues from (K) were shown in (N). Scale bar in 40× fields, 20 μm. Quantification of IHC scores (O). ( P ) Schematic representing proposed strategy to use CBX3 as a potential companion diagnostic for CK2 inhibitor to improve the sensitivity of prostate cancer to PARP inhibitor-based therapy.

Journal: Science Advances

Article Title: CK2-dependent degradation of CBX3 dictates replication fork stalling and PARP inhibitor sensitivity

doi: 10.1126/sciadv.adk8908

Figure Lengend Snippet: ( A ) Dose-response survival curves of EV or CBX3 expressing cells exposed to increasing concentrations of olaparib in PC-3 cells. ( B and C ) Colony formation assays from PC-3 cell lines infected with lentivirus expressing EV or CBX3. Representative colonies are shown in (B) with quantification data shown in (C). Scale bar, 5 mm. ( D to F ) PC-3 cells infected with lentivirus expressing EV or CBX3 were injected subcutaneously into the right flank of SCID mice and treated with vehicle or olaparib (50 mg/kg). Tumors were harvested, photographed and shown in (D). Tumor volume was measured in (E). Tumor weight was measured in (F). ( G ) WB analysis of WCL derived from PC-3 and U2OS cells treated with indicated inhibitors. ( H ) Dose-response survival curves of control or CK2 inhibitor treated cells exposed to increasing concentrations of olaparib in PC-3 cells. ( I and J ) Colony formation assays were performed in PC-3 cell lines treated with indicated inhibitor. Representative colonies were shown in (I) with quantification data shown in (J). Scale bar, 5 mm. ( K to M ) PC-3 cells were injected subcutaneously into the right flank of SCID mice and treated with vehicle, CK2 inhibitor (75 mg/kg), olaparib (50 mg/kg), or combination. Tumors in each group at day 49 were harvested, and photographed shown in (K). Tumor volume (L). Tumor weight (M). ( N and O ) Representative images of IHC staining of γ-H2AX on PC-3 tumor tissues from (K) were shown in (N). Scale bar in 40× fields, 20 μm. Quantification of IHC scores (O). ( P ) Schematic representing proposed strategy to use CBX3 as a potential companion diagnostic for CK2 inhibitor to improve the sensitivity of prostate cancer to PARP inhibitor-based therapy.

Article Snippet: Primary antibodies used were CBX3 (1:1000; Santa Cruz Biotechnology, catalog no. sc-398562), CBX1 (1:1000; Proteintech Group catalog no. 10241-2-AP), CBX5 (1:1000; Proteintech Group catalog no. 11831-1-AP), RPA2 (1:500; Cell Signaling Technology, catalog no. 35869), RPA1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-271564), RPA3 (1:1000; Cell Signaling Technology, catalog no. 42206), HA.11 (1:1000; Covance, catalog no. MMS-101R), RFWD3 (1:1000; Abcam, catalog no. ab222149), UNG2 (1:500; Santa Cruz Biotechnology, catalog no. sc-390255), SMARCAL1 (1:500; Santa Cruz Biotechnology, catalog no. sc-376377), Myc (1:1000; Santa Cruz Biotechnology, catalog no. sc-40), Flag (1:1000; Sigma-Aldrich, catalog no. F-3165), extracellular signal–regulated kinase 2 (1:1000; Santa Cruz Biotechnology, catalog no. sc-1647), phospho Histone H2A.X (S139) (1:1000; Cell Signaling Technology, catalog no. 9718), phospho Histone H2A.X (S139) (1:1000; Cell Signaling Technology, catalog no. 80312S), phospho-RPA2 (S4/S8) (1:1000; Abcam, catalog no. ab87277), phospho-RPA2 (T21) (1:1000; Abcam, catalog no. ab61065), CHK1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-8408), phospho CHK1 (S345) (1:1000; Cell Signaling Technology, catalog no. 2348), ubiquitin (1:1000; Santa Cruz Biotechnology, catalog no. sc-8017), glyceraldehyde-3-phosphate dehydrogenase (1:1000; Santa Cruz Biotechnology, catalog no. sc-47724), CDH1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-56312), CDC20 (1:1000; Santa Cruz Biotechnology, catalog no. sc-13162), and His (1:1000; Santa Cruz Biotechnology, catalog no. sc-53073).

Techniques: Expressing, Infection, Injection, Derivative Assay, Control, Immunohistochemistry, Diagnostic Assay

Under normal conditions, phosphorylation of CBX3 serine-95 by CK2 kinase augments CDH1-mediated CBX3 degradation and RPA2 dynamics at stalled replication forks, thereby promoting replication restart. However, prostate cancer–associated amplification or CK2 inhibitor–induced CBX3 high expression inhibits RFWD3 binding with RPA2, which consequently traps RPA2 at stalled replication forks, resulting in replication stress and enabling synthetic lethality to PARP inhibition.

Journal: Science Advances

Article Title: CK2-dependent degradation of CBX3 dictates replication fork stalling and PARP inhibitor sensitivity

doi: 10.1126/sciadv.adk8908

Figure Lengend Snippet: Under normal conditions, phosphorylation of CBX3 serine-95 by CK2 kinase augments CDH1-mediated CBX3 degradation and RPA2 dynamics at stalled replication forks, thereby promoting replication restart. However, prostate cancer–associated amplification or CK2 inhibitor–induced CBX3 high expression inhibits RFWD3 binding with RPA2, which consequently traps RPA2 at stalled replication forks, resulting in replication stress and enabling synthetic lethality to PARP inhibition.

Article Snippet: Primary antibodies used were CBX3 (1:1000; Santa Cruz Biotechnology, catalog no. sc-398562), CBX1 (1:1000; Proteintech Group catalog no. 10241-2-AP), CBX5 (1:1000; Proteintech Group catalog no. 11831-1-AP), RPA2 (1:500; Cell Signaling Technology, catalog no. 35869), RPA1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-271564), RPA3 (1:1000; Cell Signaling Technology, catalog no. 42206), HA.11 (1:1000; Covance, catalog no. MMS-101R), RFWD3 (1:1000; Abcam, catalog no. ab222149), UNG2 (1:500; Santa Cruz Biotechnology, catalog no. sc-390255), SMARCAL1 (1:500; Santa Cruz Biotechnology, catalog no. sc-376377), Myc (1:1000; Santa Cruz Biotechnology, catalog no. sc-40), Flag (1:1000; Sigma-Aldrich, catalog no. F-3165), extracellular signal–regulated kinase 2 (1:1000; Santa Cruz Biotechnology, catalog no. sc-1647), phospho Histone H2A.X (S139) (1:1000; Cell Signaling Technology, catalog no. 9718), phospho Histone H2A.X (S139) (1:1000; Cell Signaling Technology, catalog no. 80312S), phospho-RPA2 (S4/S8) (1:1000; Abcam, catalog no. ab87277), phospho-RPA2 (T21) (1:1000; Abcam, catalog no. ab61065), CHK1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-8408), phospho CHK1 (S345) (1:1000; Cell Signaling Technology, catalog no. 2348), ubiquitin (1:1000; Santa Cruz Biotechnology, catalog no. sc-8017), glyceraldehyde-3-phosphate dehydrogenase (1:1000; Santa Cruz Biotechnology, catalog no. sc-47724), CDH1 (1:1000; Santa Cruz Biotechnology, catalog no. sc-56312), CDC20 (1:1000; Santa Cruz Biotechnology, catalog no. sc-13162), and His (1:1000; Santa Cruz Biotechnology, catalog no. sc-53073).

Techniques: Phospho-proteomics, Amplification, Expressing, Binding Assay, Inhibition

Persistent hyperemic foci exhibit evidence of dermal senescence. ( A – E ) Dermal cells in areas of high Hgb content exhibit a heterochromatin pattern of DAPI staining. After excision of skin from mice treated with and without UV at 2 and 20 weeks after stopping UV treatments, the sections were stained with DAPI to better demonstrate nuclear morphology. Nuclei of dermal cells showing a heterochromatin staining pattern are shown by white arrows while nuclei exhibiting a euchromatin pattern of DAPI staining are shown by yellow arrows. The hatched white line outlines the epidermal-dermal junction. The white scale bar represents 50 µm. ( A ) Skin from an area of low Hgb content 2 weeks after stopping UV treatments. ( B ) Skin from an area of high Hgb content 2 weeks after stopping UV treatments. ( C ) Control non-UV treated skin. ( D ) Skin from an area of low Hgb content 20 weeks after stopping UV. ( E ) Skin from an area of high Hgb content 20 weeks after stopping UV. (F – J ) Representative photomigrographs of skin following immunolabeling with anti-p16 INK4a antibodies. ( F ) Control non-UV treated epidermis. ( G , H ) Skin excised 2 weeks after stopping UV treatments from an area of low Hgb content ( G ) or from an area of high Hgb content ( H ). ( I , J ) Skin excised from a low Hgb area ( I ) or a high Hgb area ( J ) at 20 weeks after stopping UV treatments. Black arrows in ( H , J ) show dermal cells with enlarged nuclei labeling positive for p16 INK4a . The black scale bars represent 100 µm. ( K ) HP1γ + dermal cells are increased in hyperemic areas at both 2 and 20 weeks after stopping UV treatments. Immunofluorescent (IF) labeling of formalin-fixed skin sections was performed using anti-HP1γ and anti-pancytokeratin (CK) antibody. The data depicts the % of dermal cells positive for HP1γ nuclear labeling. ( L ) Dermal cells positive for nuclear γH2AX are also increased in hyperemic areas only at 2 and 20 weeks post-UV. IF was performed for both nuclear γH2AX immunolabeling and CK. The data shown is the percentage of γH2AX + dermal cells relative to all CK negative cells. (ns = non-significant; ** = p < 0.01; *** = p < 0.001).

Journal: Scientific Reports

Article Title: Evidence for a non-stochastic two-field hypothesis for persistent skin cancer risk

doi: 10.1038/s41598-020-75864-2

Figure Lengend Snippet: Persistent hyperemic foci exhibit evidence of dermal senescence. ( A – E ) Dermal cells in areas of high Hgb content exhibit a heterochromatin pattern of DAPI staining. After excision of skin from mice treated with and without UV at 2 and 20 weeks after stopping UV treatments, the sections were stained with DAPI to better demonstrate nuclear morphology. Nuclei of dermal cells showing a heterochromatin staining pattern are shown by white arrows while nuclei exhibiting a euchromatin pattern of DAPI staining are shown by yellow arrows. The hatched white line outlines the epidermal-dermal junction. The white scale bar represents 50 µm. ( A ) Skin from an area of low Hgb content 2 weeks after stopping UV treatments. ( B ) Skin from an area of high Hgb content 2 weeks after stopping UV treatments. ( C ) Control non-UV treated skin. ( D ) Skin from an area of low Hgb content 20 weeks after stopping UV. ( E ) Skin from an area of high Hgb content 20 weeks after stopping UV. (F – J ) Representative photomigrographs of skin following immunolabeling with anti-p16 INK4a antibodies. ( F ) Control non-UV treated epidermis. ( G , H ) Skin excised 2 weeks after stopping UV treatments from an area of low Hgb content ( G ) or from an area of high Hgb content ( H ). ( I , J ) Skin excised from a low Hgb area ( I ) or a high Hgb area ( J ) at 20 weeks after stopping UV treatments. Black arrows in ( H , J ) show dermal cells with enlarged nuclei labeling positive for p16 INK4a . The black scale bars represent 100 µm. ( K ) HP1γ + dermal cells are increased in hyperemic areas at both 2 and 20 weeks after stopping UV treatments. Immunofluorescent (IF) labeling of formalin-fixed skin sections was performed using anti-HP1γ and anti-pancytokeratin (CK) antibody. The data depicts the % of dermal cells positive for HP1γ nuclear labeling. ( L ) Dermal cells positive for nuclear γH2AX are also increased in hyperemic areas only at 2 and 20 weeks post-UV. IF was performed for both nuclear γH2AX immunolabeling and CK. The data shown is the percentage of γH2AX + dermal cells relative to all CK negative cells. (ns = non-significant; ** = p < 0.01; *** = p < 0.001).

Article Snippet: Immunofluorescent staining of formalin-fixed paraffin-embedded mouse skin following heat-induced antigen retrieval was performed using the following antibodies: HP1γ (Cbx3) immunolabeling was done using rabbit anti-Cbx3 (1:250, Cat#IHC-00204, Bethyl Laboratories). γH2AX immunolabeling was done using monoclonal rabbit anti-γH2AX (1:100, Bethyl Laboratories, Cat#A700-053).

Techniques: Staining, Control, Immunolabeling, Labeling

Persistent hyperemic foci exhibit evidence of dermal senescence. ( A – E ) Dermal cells in areas of high Hgb content exhibit a heterochromatin pattern of DAPI staining. After excision of skin from mice treated with and without UV at 2 and 20 weeks after stopping UV treatments, the sections were stained with DAPI to better demonstrate nuclear morphology. Nuclei of dermal cells showing a heterochromatin staining pattern are shown by white arrows while nuclei exhibiting a euchromatin pattern of DAPI staining are shown by yellow arrows. The hatched white line outlines the epidermal-dermal junction. The white scale bar represents 50 µm. ( A ) Skin from an area of low Hgb content 2 weeks after stopping UV treatments. ( B ) Skin from an area of high Hgb content 2 weeks after stopping UV treatments. ( C ) Control non-UV treated skin. ( D ) Skin from an area of low Hgb content 20 weeks after stopping UV. ( E ) Skin from an area of high Hgb content 20 weeks after stopping UV. (F – J ) Representative photomigrographs of skin following immunolabeling with anti-p16 INK4a antibodies. ( F ) Control non-UV treated epidermis. ( G , H ) Skin excised 2 weeks after stopping UV treatments from an area of low Hgb content ( G ) or from an area of high Hgb content ( H ). ( I , J ) Skin excised from a low Hgb area ( I ) or a high Hgb area ( J ) at 20 weeks after stopping UV treatments. Black arrows in ( H , J ) show dermal cells with enlarged nuclei labeling positive for p16 INK4a . The black scale bars represent 100 µm. ( K ) HP1γ + dermal cells are increased in hyperemic areas at both 2 and 20 weeks after stopping UV treatments. Immunofluorescent (IF) labeling of formalin-fixed skin sections was performed using anti-HP1γ and anti-pancytokeratin (CK) antibody. The data depicts the % of dermal cells positive for HP1γ nuclear labeling. ( L ) Dermal cells positive for nuclear γH2AX are also increased in hyperemic areas only at 2 and 20 weeks post-UV. IF was performed for both nuclear γH2AX immunolabeling and CK. The data shown is the percentage of γH2AX + dermal cells relative to all CK negative cells. (ns = non-significant; ** = p < 0.01; *** = p < 0.001).

Journal:

Article Title:

doi:

Figure Lengend Snippet: Persistent hyperemic foci exhibit evidence of dermal senescence. ( A – E ) Dermal cells in areas of high Hgb content exhibit a heterochromatin pattern of DAPI staining. After excision of skin from mice treated with and without UV at 2 and 20 weeks after stopping UV treatments, the sections were stained with DAPI to better demonstrate nuclear morphology. Nuclei of dermal cells showing a heterochromatin staining pattern are shown by white arrows while nuclei exhibiting a euchromatin pattern of DAPI staining are shown by yellow arrows. The hatched white line outlines the epidermal-dermal junction. The white scale bar represents 50 µm. ( A ) Skin from an area of low Hgb content 2 weeks after stopping UV treatments. ( B ) Skin from an area of high Hgb content 2 weeks after stopping UV treatments. ( C ) Control non-UV treated skin. ( D ) Skin from an area of low Hgb content 20 weeks after stopping UV. ( E ) Skin from an area of high Hgb content 20 weeks after stopping UV. (F – J ) Representative photomigrographs of skin following immunolabeling with anti-p16 INK4a antibodies. ( F ) Control non-UV treated epidermis. ( G , H ) Skin excised 2 weeks after stopping UV treatments from an area of low Hgb content ( G ) or from an area of high Hgb content ( H ). ( I , J ) Skin excised from a low Hgb area ( I ) or a high Hgb area ( J ) at 20 weeks after stopping UV treatments. Black arrows in ( H , J ) show dermal cells with enlarged nuclei labeling positive for p16 INK4a . The black scale bars represent 100 µm. ( K ) HP1γ + dermal cells are increased in hyperemic areas at both 2 and 20 weeks after stopping UV treatments. Immunofluorescent (IF) labeling of formalin-fixed skin sections was performed using anti-HP1γ and anti-pancytokeratin (CK) antibody. The data depicts the % of dermal cells positive for HP1γ nuclear labeling. ( L ) Dermal cells positive for nuclear γH2AX are also increased in hyperemic areas only at 2 and 20 weeks post-UV. IF was performed for both nuclear γH2AX immunolabeling and CK. The data shown is the percentage of γH2AX + dermal cells relative to all CK negative cells. (ns = non-significant; ** = p < 0.01; *** = p < 0.001).

Article Snippet: Immunofluorescent staining of formalin-fixed paraffin-embedded mouse skin following heat-induced antigen retrieval was performed using the following antibodies: HP1γ (Cbx3) immunolabeling was done using rabbit anti-Cbx3 (1:250, Cat#IHC-00204, Bethyl Laboratories). γH2AX immunolabeling was done using monoclonal rabbit anti-γH2AX (1:100, Bethyl Laboratories, Cat#A700-053).

Techniques: Staining, Immunolabeling, Labeling

Figure 1. Identification of MC antigens reactive to IgA auto-Abs generated in gddY mice. (A) Detection of antigens for rmAbs derived from gddY mouse PBs in the lysates of gddY mouse primary MCs by WB. Split membranes, each containing proteins in one lane, were incubated with the indicated rmAbs (1 μg/ml), followed by an anti-human IgG Ab. (B) Immunoprecipitation of gddY mouse MC lysates with rmAb#66 or the control rmAb#NP. SDS–PAGE of the precipitates was developed using silver staining. The bands indicated by arrows were excised and analyzed using mass spectrometry. Those from the rmAb#66 precipitate included CBX1, CBX3, and CBX5, as indicated by arrows. (C) FLAG-tagged CBX1, CBX3, or CBX5 proteins transiently expressed in HEK293T cells were detected by WB with the indicated rmAbs (1 μg/ml), followed by anti-human IgG Ab (left panel) or anti-FLAG Ab (right panel). (D) ELISA screening of rmAbs for binding to CBX proteins. The heatmap represents the resulting OD values of the indicated rmAbs, with “+” indicating a saturated signal. (E) Reactivity of gddY mouse serum IgA with the CBX proteins. WB was performed as in (C) using sera of individual BALB/c or gddY mice as the primary Ab as indicated and anti-IgA Ab as the secondary Ab (left, top) or anti-FLAG Ab to confirm the presence of CBX proteins on the blot (left, bottom). Representative data are shown here. Venn diagram showing the percentage of gddY mice whose serum IgA reacted with CBX1, CBX3, or CBX5 (right panel) (n = 14). (F) ELISA determination of anti-CBX3 IgA Abs in sera from healthy controls (HC; n = 30) and patients with IgAN (IgAN; n = 70). ***: 0.0006 (Mann–Whitney U test). The dashed line shows 99% confidence interval (CI) of the control sera. One of two or three independent experiments with similar results is shown in each panel. Source data are available for this figure.

Journal: Life science alliance

Article Title: Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

doi: 10.26508/lsa.202402588

Figure Lengend Snippet: Figure 1. Identification of MC antigens reactive to IgA auto-Abs generated in gddY mice. (A) Detection of antigens for rmAbs derived from gddY mouse PBs in the lysates of gddY mouse primary MCs by WB. Split membranes, each containing proteins in one lane, were incubated with the indicated rmAbs (1 μg/ml), followed by an anti-human IgG Ab. (B) Immunoprecipitation of gddY mouse MC lysates with rmAb#66 or the control rmAb#NP. SDS–PAGE of the precipitates was developed using silver staining. The bands indicated by arrows were excised and analyzed using mass spectrometry. Those from the rmAb#66 precipitate included CBX1, CBX3, and CBX5, as indicated by arrows. (C) FLAG-tagged CBX1, CBX3, or CBX5 proteins transiently expressed in HEK293T cells were detected by WB with the indicated rmAbs (1 μg/ml), followed by anti-human IgG Ab (left panel) or anti-FLAG Ab (right panel). (D) ELISA screening of rmAbs for binding to CBX proteins. The heatmap represents the resulting OD values of the indicated rmAbs, with “+” indicating a saturated signal. (E) Reactivity of gddY mouse serum IgA with the CBX proteins. WB was performed as in (C) using sera of individual BALB/c or gddY mice as the primary Ab as indicated and anti-IgA Ab as the secondary Ab (left, top) or anti-FLAG Ab to confirm the presence of CBX proteins on the blot (left, bottom). Representative data are shown here. Venn diagram showing the percentage of gddY mice whose serum IgA reacted with CBX1, CBX3, or CBX5 (right panel) (n = 14). (F) ELISA determination of anti-CBX3 IgA Abs in sera from healthy controls (HC; n = 30) and patients with IgAN (IgAN; n = 70). ***: 0.0006 (Mann–Whitney U test). The dashed line shows 99% confidence interval (CI) of the control sera. One of two or three independent experiments with similar results is shown in each panel. Source data are available for this figure.

Article Snippet: For FCM, the following Abs were used: anti-mouse IgA-APC (clone 11- 44-2; Southern Biotech), anti-mouse CD73-biotin (clone TY/11.8; BioLegend), anti-mouse CD45-PerCP-Cy5.5 (clone 30-F11; BioLegend), anti-mouse CD31-PE (clone 390; BioLegend), anti-mouse TER119BV510 (clone TER-119; BioLegend), rabbit polyclonal anti-HP1γ (CBX3) Ab (2619; Cell Signaling Technology), rabbit monoclonal anti-HP1β (CBX1) Ab (8676; Cell Signaling Technology), rabbit IgG (for isotypematched control; 011-000-003; Jackson ImmunoResearch), antirabbit IgG-BV421 (clone Poly4064; BioLegend), and anti-human IgG Fcγ-Alexa Fluor 647 (709-606-098; Jackson ImmunoResearch).

Techniques: Generated, Derivative Assay, Incubation, Immunoprecipitation, Control, SDS Page, Silver Staining, Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Binding Assay, MANN-WHITNEY

Figure 6. Identification of a bacterial strain that shares an rmAb#66 epitope with CBX3. (A) Isolated bacteria (C42) were stained with rmAb#66 or rmAb#NP (1 μg/ml), followed by Alexa Fluor 647–anti-human IgG together with SYBR Green, and analyzed by FCM. (B) rmAb#66 (1 μg/ml) was incubated with CBX3 or OVA and then with C42, which was stained with anti-human IgG and analyzed as described in Fig 5G. Data are presented as means ± SD of two technical replicates. (C) C42 bacteria (2 × 106 cells) were incubated with the indicated concentrations of PNGase F (Roche) for 4 h at 37°C, stained with rmAb#66 (1 μg/ml) overnight on ice, and labeled with SYBR Green. Binding of rmAb#66 to C42 was detected by FCM using Alexa Fluor 647-anti-human IgG (n = 2). (D) BALB/c mice were immunized s.c. with UV-killed C42 or E. faecalis, together with zymosan as an adjuvant, at 4 wk of age. WB analysis was performed as shown in Fig 3E (except that CBX1 and CBX5 samples were omitted) with sera from immunized mice at 8 wk of age as the primary Abs and anti-IgG Ab as the secondary Ab (left panel) or anti-FLAG Ab (right panel). (E) Representative IFM images of kidney sections from the mice used in (D). Kidney sections were stained with an anti-IgA Ab (red). Dashed circles indicate areas of glomeruli and white lines indicate scale bars (100 μm). (F) Frequency of bacteria recognized by rmAb#66 among all bacteria (SYBR Green+) in the oral cavity of gddY mice (n = 6). Bacteria from gddY mice fed a normal diet (control) or an ED (ED) were cultured anaerobically on brain heart infusion agar, stained with rmAb#66 (1 μg/ml) and Alexa Fluor 647–anti-human IgG, together with SYBR Green, and analyzed by FCM. (G) Frequency of each bacterial strain among the total bacteria in the oral cavities of gddY mice fed a normal diet (control) or an ED, as determined by 16S rRNA bulk sequencing (n = 6). An underscore after each genus name indicates that the species was not classified. Data are presented as means ± SD of biological replicates. *P < 0.05; ***P < 0.005; ****P < 0.001. P-values were calculated using the unpaired t test. One of two independent experiments with similar results is shown in each panel. Source data are available for this figure.

Journal: Life science alliance

Article Title: Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

doi: 10.26508/lsa.202402588

Figure Lengend Snippet: Figure 6. Identification of a bacterial strain that shares an rmAb#66 epitope with CBX3. (A) Isolated bacteria (C42) were stained with rmAb#66 or rmAb#NP (1 μg/ml), followed by Alexa Fluor 647–anti-human IgG together with SYBR Green, and analyzed by FCM. (B) rmAb#66 (1 μg/ml) was incubated with CBX3 or OVA and then with C42, which was stained with anti-human IgG and analyzed as described in Fig 5G. Data are presented as means ± SD of two technical replicates. (C) C42 bacteria (2 × 106 cells) were incubated with the indicated concentrations of PNGase F (Roche) for 4 h at 37°C, stained with rmAb#66 (1 μg/ml) overnight on ice, and labeled with SYBR Green. Binding of rmAb#66 to C42 was detected by FCM using Alexa Fluor 647-anti-human IgG (n = 2). (D) BALB/c mice were immunized s.c. with UV-killed C42 or E. faecalis, together with zymosan as an adjuvant, at 4 wk of age. WB analysis was performed as shown in Fig 3E (except that CBX1 and CBX5 samples were omitted) with sera from immunized mice at 8 wk of age as the primary Abs and anti-IgG Ab as the secondary Ab (left panel) or anti-FLAG Ab (right panel). (E) Representative IFM images of kidney sections from the mice used in (D). Kidney sections were stained with an anti-IgA Ab (red). Dashed circles indicate areas of glomeruli and white lines indicate scale bars (100 μm). (F) Frequency of bacteria recognized by rmAb#66 among all bacteria (SYBR Green+) in the oral cavity of gddY mice (n = 6). Bacteria from gddY mice fed a normal diet (control) or an ED (ED) were cultured anaerobically on brain heart infusion agar, stained with rmAb#66 (1 μg/ml) and Alexa Fluor 647–anti-human IgG, together with SYBR Green, and analyzed by FCM. (G) Frequency of each bacterial strain among the total bacteria in the oral cavities of gddY mice fed a normal diet (control) or an ED, as determined by 16S rRNA bulk sequencing (n = 6). An underscore after each genus name indicates that the species was not classified. Data are presented as means ± SD of biological replicates. *P < 0.05; ***P < 0.005; ****P < 0.001. P-values were calculated using the unpaired t test. One of two independent experiments with similar results is shown in each panel. Source data are available for this figure.

Article Snippet: For FCM, the following Abs were used: anti-mouse IgA-APC (clone 11- 44-2; Southern Biotech), anti-mouse CD73-biotin (clone TY/11.8; BioLegend), anti-mouse CD45-PerCP-Cy5.5 (clone 30-F11; BioLegend), anti-mouse CD31-PE (clone 390; BioLegend), anti-mouse TER119BV510 (clone TER-119; BioLegend), rabbit polyclonal anti-HP1γ (CBX3) Ab (2619; Cell Signaling Technology), rabbit monoclonal anti-HP1β (CBX1) Ab (8676; Cell Signaling Technology), rabbit IgG (for isotypematched control; 011-000-003; Jackson ImmunoResearch), antirabbit IgG-BV421 (clone Poly4064; BioLegend), and anti-human IgG Fcγ-Alexa Fluor 647 (709-606-098; Jackson ImmunoResearch).

Techniques: Isolation, Bacteria, Staining, SYBR Green Assay, Incubation, Labeling, Binding Assay, Adjuvant, Control, Cell Culture, Sequencing