Anti-UBN1 antibody

• WB

Unknown

Western blot - Anti-UBN1 antibody (AB101282)

All lanes:

Western blot - Anti-UBN1 antibody (ab101282) at 0.1 µg/mL

Lane 1:

HeLa whole cell lysate at 50 µg

Lane 2:

HeLa whole cell lysate at 15 µg

Lane 3:

HeLa whole cell lysate at 5 µg

Lane 4:

293T whole cell lysate at 50 µg

Predicted band size: 122 kDa

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Exposure time: 3min

• WB

CiteAb

Western blot - Anti-UBN1 antibody (AB101282)

Western Blotting using Anti-UBN1 antibody, ab101282. Publication image from Ray-Gallet, D. et al., 2018, Nat Commun, 30082790. Legend direct from paper.

HIRA homooligomerizes in cells and forms a homotrimer in vitro. a YFP constructs of human HIRA and mutants. The WD40 repeat (aa 1–369), B (aa 439–475) and C (aa 763–963) domains involved in UBN, ASF1a, and CABIN1 interactions, respectively, are shown. Star indicates single amino acid substitution with alanine. The co-immunoprecipitation efficiency of endogenous HIRA or HIRA-HA (HIRA coIP) is indicated for each construct, “+” indicates that the efficiency of coIP is similar to the one obtained with the wt HIRA construct and “−” indicates that the efficiency of the coIP is decreased. b Western blot analysis of anti-GFP-immunoprecipitates from U2OS nuclear extracts expressing YFP-tagged proteins. c Western blot analysis of anti-GFP-immunoprecipitates from U2OS nuclear extracts expressing both YFP-tagged and HIRA-HA proteins. d Western blot analysis of anti-GFP-immunoprecipitates from U2OS nuclear extracts expressing YFP-tagged proteins. In b, c and d, input corresponds to 10% of nuclear extract used for each experiment. e Equilibrium sedimentation of recombinant proteins HIRA(661–1017), CABIN1 full length (FL) and HIRA(661–1017) + CABIN1 FL. Theoretical (open symbols) and experimental (closed symbol) curves are shown

false

• WB

CiteAb

Western blot - Anti-UBN1 antibody (AB101282)

Western Blotting using Anti-UBN1 antibody, ab101282. Publication image from Ray-Gallet, D. et al., 2018, Nat Commun, 30082790. Legend direct from paper.

HIRA homooligomerization is required for CABIN1 interaction. a YFP constructs of human HIRA and mutants. The amino acids R227 and I461 critical for UBN and ASF1a interactions, respectively, are shown. Star indicates single amino acid substitution (R227 with K or I461 with D). The co-immunoprecipitation efficiencies of both endogenous HIRA (HIRA coIP) and CABIN1 (CABIN1 coIP) are indicated for each construct, “+” indicates that the efficiency of coIP is similar to the one obtained with the wt HIRA construct and “−” indicates that the efficiency of the coIP is decreased. b Western blot analysis of anti-GFP-immunoprecipitates from U2OS nuclear extracts expressing YFP-tagged proteins. c Western blot analysis of anti-GFP-immunoprecipitates from U2OS nuclear extracts expressing both HIRA-YFP and HIRA-HA proteins prepared from cells treated with siRNAs control or CABIN1. In b and c, input corresponds to 10% of nuclear extract used for each experiment

false

• WB

CiteAb

Western blot - Anti-UBN1 antibody (AB101282)

Western Blotting using Anti-UBN1 antibody, ab101282. Publication image from Ray-Gallet, D. et al., 2018, Nat Commun, 30082790. Legend direct from paper.

HIRA homooligomerization is required for CABIN1 interaction. a YFP constructs of human HIRA and mutants. The amino acids R227 and I461 critical for UBN and ASF1a interactions, respectively, are shown. Star indicates single amino acid substitution (R227 with K or I461 with D). The co-immunoprecipitation efficiencies of both endogenous HIRA (HIRA coIP) and CABIN1 (CABIN1 coIP) are indicated for each construct, “+” indicates that the efficiency of coIP is similar to the one obtained with the wt HIRA construct and “−” indicates that the efficiency of the coIP is decreased. b Western blot analysis of anti-GFP-immunoprecipitates from U2OS nuclear extracts expressing YFP-tagged proteins. c Western blot analysis of anti-GFP-immunoprecipitates from U2OS nuclear extracts expressing both HIRA-YFP and HIRA-HA proteins prepared from cells treated with siRNAs control or CABIN1. In b and c, input corresponds to 10% of nuclear extract used for each experiment

false

• WB

CiteAb

Western blot - Anti-UBN1 antibody (AB101282)

Western Blotting using Anti-UBN1 antibody, ab101282. Publication image from Ray-Gallet, D. et al., 2018, Nat Commun, 30082790. Legend direct from paper.

Homotrimerization of HIRA is critical for its enrichment at UV damaged sites and for new H3.3 deposition. a (Left, top) Scheme of the experimental assay for HIRA-YFP enrichment at local UV damage in U2OS cells. (Left, bottom) The graph shows the percentage of U2OS cells with HIRA-YFP protein colocalizing with XPB foci. Error bars represent SD from three independent experiments. Statistical analysis using a t-test was performed with Prism 7 software (ns : nonsignificant, *p < 0.05, ***p < 0.001). (Right) Images of representative U2OS cells irradiated locally with UV exhibiting an enrichment of HIRA wt or an absence of enrichment of HIRA (W799A–D800A) at one damage site identified by anti-XPB immunofluorescence. Scale bar 5 µm. b (Top, left) Scheme of the experimental assay for new H3.3 deposition by a Quench-Chase-Pulse experiment (QCP) in HeLa H3.3-SNAP-HA HIRA KO cells transfected with HIRA-HA constructs. (Top, right) Western blot analysis showing the protein expression of exogeneous HIRA-HA wt and (W799A–D800A) mutant. (Bottom, left) Images of cells subjected to QCP : representative images for a cell non-transfected or transfected with HIRA-HA (W799A–D800A) mutant and a high TMR positive representative image for a cell transfected with HIRA-HA wt. (Bottom, right). One graph shows the fluorescence intensity ratio TMR/TMR non-transfected. The other graph gives the percentage of cells exhibiting a positive profile for H3.3 deposition. Error bars represent the SD from three independent experiments. Statistical analysis using a t-test was performed with Prism 7 software (ns : nonsignificant, *p < 0.05)

false

• WB

CiteAb

Western blot - Anti-UBN1 antibody (AB101282)

Western Blotting using Anti-UBN1 antibody, ab101282. Publication image from Ray-Gallet, D. et al., 2018, Nat Commun, 30082790. Legend direct from paper.

Domains of HIRA involved in its homooligomerization and CABIN1 interaction are distinct. a YFP constructs of human HIRA and mutants. Three different structures matching the previously described C domain are indicated : β-strand (aa 661–872), loop (aa 873–904) andα-helical (aa 905–1017) domains. Star indicates single amino acid substitution with alanine. The co-immunoprecipitation efficiency of endogenous CABIN1 (CABIN1 coIP) is indicated for each construct, “+” indicates that the efficiency of coIP is similar to the one obtained with the wt HIRA construct and “−” indicates that the efficiency of the coIP is decreased. b Western blot analysis of anti-GFP-immunoprecipitates from U2OS nuclear extracts expressing YFP-tagged proteins. Input corresponds to 10% of nuclear extract used for each experiment. c (Left) Western blot analysis of nuclear extracts from HIRA KO and control HeLa cells. (Right) Western blot analysis of nuclear extracts from HeLa HIRA KO cells expressing YFP-tagged proteins. d Superose 6 fractionation of recombinant proteins CABIN1 FL, CABIN1 FL + HIRA(661–1017) and CABIN1 FL + HIRA(661–1017) δ873–904. The dashes indicate the fractions in which free CABIN1 FL elutes. Input corresponds to protein at the concentration that it was loaded onto the Superose 6 column. e Equilibrium sedimentation of recombinant protein HIRA(661–1017) δ873–904. Theoretical (open symbols) and experimental (closed symbol) curves are shown

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