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1.
FIG. 5.

FIG. 5. From: Human Rvb1/Tip49 Is Required for the Histone Acetyltransferase Activity of Tip60/NuA4 and for the Downregulation of Phosphorylation on H2AX after DNA Damage .

The depletion of either Ino80 or SRCAP does not affect levels of phospho-H2AX after DNA damage. (A) The depletion of Ino80 does not increase total phospho-H2AX levels after DNA damage. HeLa cells transfected with the indicated siRNAs were immunoblotted for total phospho-H2AX and H2AX protein. A decrease in Ino80 was detected by using anti-Ino80 antibody, whereas β-actin is shown as a loading control. Cells were treated with UV 72 h after transfection with siRNAs and harvested 60 min postirradiation. H2AXpS139, H2AX phosphorylated on S139; +, present. (B) The knockdown of SRCAP does not increase total phospho-H2AX levels after DNA damage. HeLa cells transfected with the indicated siRNAs were immunoblotted for total phospho-H2AX and H2AX. The depletion of SRCAP was detected by using anti-SRCAP antibody. The experiment was carried out as described for panel A.

Sudhakar Jha, et al. Mol Cell Biol. 2008 April;28(8):2690-2700.
2.
FIG. 4.

FIG. 4. From: Human Rvb1/Tip49 Is Required for the Histone Acetyltransferase Activity of Tip60/NuA4 and for the Downregulation of Phosphorylation on H2AX after DNA Damage .

Rvb1 is required for the removal of phospho-H2AX from chromatin. (A) Increase of phospho-H2AX after Rvb1 and PP2A depletion. Cell lysates prepared after the depletion of Rvb1 and PP2A alone or together were immunoblotted with the indicated antibodies. H2AXpS139, H2AX phosphorylated on S139; +, present; −, absent. (B) Quantitation of phospho-H2AX signals in panel A by using Scion imaging software. (C) Increased phospho-H2AX was present on chromatin after the knockdown of Rvb1. The phospho-H2AX foci seen in cells depleted of Rvb1 were bound to chromatin. HeLa cells transfected with the indicated siRNAs with (+) or without (−) UV irradiation were extracted in situ to remove non-chromatin-bound proteins prior to fixation and immunostaining for phospho-H2AX. (D) The loss of PP2A stabilizes phospho-H2AX in the soluble fraction, and this effect is dependent on Rvb1. HeLa cells transfected with the indicated siRNAs were fractionated 60 min after UV irradiation. The fractions were immunoblotted for the indicated proteins. *, cross-reacting band.

Sudhakar Jha, et al. Mol Cell Biol. 2008 April;28(8):2690-2700.
3.
FIG. 6.

FIG. 6. From: Human Rvb1/Tip49 Is Required for the Histone Acetyltransferase Activity of Tip60/NuA4 and for the Downregulation of Phosphorylation on H2AX after DNA Damage .

The depletion of Tip60 increases phospho-H2AX. (A) The depletion of Tip60 increases total phospho-H2AX levels after DNA damage. HeLa cells transfected with the indicated siRNAs with or without (−) UV irradiation were immunoblotted as shown. H2AXpS139, H2AX phosphorylated on S139; +, present. (B) Immunofluorescence after Tip60 depletion. HeLa cells transfected with the indicated siRNAs with or without UV irradiation were extracted in situ to remove non-chromatin-bound proteins prior to fixation and immunostaining for phospho-H2AX. (C) Percentages of cells positive for phospho-H2AX foci in the analysis presented in panel B. 0, no UV treatment. Means ± SD of results from three experiments are shown. (D) The codepletion of Rvb1 and Tip60 does not further increase phospho-H2AX levels after UV irradiation. UV-treated HeLa cells were depleted of Rvb1 and Tip60 either individually or together by using the indicated siRNAs. Western blotting analysis with anti-phospho-H2AX, anti-H2AX, anti-Rvb1, anti-Tip60, and anti-β-actin is shown. (E) Quantitation of phospho-H2AX signals in panel D (upper lane) using Scion imaging software.

Sudhakar Jha, et al. Mol Cell Biol. 2008 April;28(8):2690-2700.
4.
FIG. 2.

FIG. 2. From: Human Rvb1/Tip49 Is Required for the Histone Acetyltransferase Activity of Tip60/NuA4 and for the Downregulation of Phosphorylation on H2AX after DNA Damage .

The Rvb1 knockdown phenotype can be reversed by the expression of exogenous Rvb1 protein. (A) The depletion of endogenous Rvb1 by siRVB1-A and siRVB1-B increases phospho-H2AX. HeLa cell lysates were prepared 72 h after the transfection of cells with the indicated siRNAs. The decrease in total Rvb1 protein was detected using anti-Rvb1 antibody, and anti-β-actin was used as a loading control. +, present; H2AXpS139, H2AX phosphorylated on S139. (B) Exogenous Flag-Rvb1 was knocked down by siRVB1-A but not siRVB1-B. HeLa cells stably expressing Flag-Rvb1 were lysed after transfection with the indicated siRNAs with (+) or without (−) UV irradiation. The experiment was carried out as described for panel A. (C) Phospho-H2AX levels after Rvb1 are restored by expressing Flag-Rvb1 resistant to siRVB1-B. HeLa cells stably expressing Flag-Rvb1 were transfected for 72 h with the indicated siRNAs before UV irradiation and immunoblotting of lysates with the indicated antibodies. (D) Phospho-H2AX foci detected by immunofluorescence. HeLa cells with or without Flag-Rvb1 were transfected with the indicated siRNAs for 72 h and either left untreated or irradiated with UV. +, present; −, absent. (E) Quantitation of phospho-H2AX foci. The percentages of cells positive for phospho-H2AX foci among cells transfected with the indicated siRNAs and those with (+UV) and without (−UV) DNA damage in the analysis presented in panel D are shown.

Sudhakar Jha, et al. Mol Cell Biol. 2008 April;28(8):2690-2700.
5.
FIG. 1.

FIG. 1. From: Human Rvb1/Tip49 Is Required for the Histone Acetyltransferase Activity of Tip60/NuA4 and for the Downregulation of Phosphorylation on H2AX after DNA Damage .

The depletion of Rvb1 increases phospho-H2AX after DNA damage. (A) Depletion of Rvb1 protein using siRNA against RVB1. Lysates prepared from HeLa cells transfected with the indicated siRNAs at the indicated times were immunoblotted for Rvb1 protein and β-actin. (B) Semiquantitative Western blot analysis of Rvb1. The indicated amounts of HeLa cell lysates were loaded and blotted with anti-Rvb1 and anti-β-actin antibodies. One hundred percent equals 20 μg of protein loaded. (C) Increase in total phospho-H2AX after DNA damage. Lysates from HeLa cells were prepared after the cells were transfected with siRNA with or without UV. UV irradiation was carried out 72 h after the transfection of cells with siRNA, and cells were harvested 60 min post-UV irradiation. Results from immunoblotting with anti-phospho-H2AX, anti-H2AX, anti-Rvb1, and anti-β-actin antibodies are shown. +, present; −, absent; H2AXpS139, H2AX phosphorylated on S139. (D) Immunofluorescence analysis with anti-phospho-H2AX antibody. HeLa cells were transfected with the indicated siRNAs and processed for immunofluorescence by using anti-phospho-H2AX antibody. DNA damage was induced as mentioned in the legend to panel C. (E) Quantitation of phospho-H2AX foci. The percentages of cells positive for phospho-H2AX foci among cells transfected with the indicated siRNAs and those with (UV) and without (0) DNA damage in the analysis presented in panel D are shown. The experiment was repeated three times, and the means ± standard deviations (SD) are plotted. (F) The loss of Rvb1 results in the persistence of phospho-H2AX. HeLa cells were transfected with the indicated siRNAs. Cells were harvested at the indicated time points after UV irradiation, and lysates were immunoblotted with the indicated antibodies.

Sudhakar Jha, et al. Mol Cell Biol. 2008 April;28(8):2690-2700.
6.
FIG. 3.

FIG. 3. From: Human Rvb1/Tip49 Is Required for the Histone Acetyltransferase Activity of Tip60/NuA4 and for the Downregulation of Phosphorylation on H2AX after DNA Damage .

Rvb1 colocalizes and interacts with phospho-H2AX after DNA damage. (A) The loss of Rvb1 does not hyperactivate ATM and ATR kinases. Lysates from HCT116 cells were prepared after the transfection of the cells with the indicated siRNAs and the harvesting of the cells at the indicated time points post-UV irradiation. The results of immunoblotting with antibodies to Chk1 phosphorylated on S317 (Chk1pS317) and Chk phosphorylated on T68 (Chk2pT68) and anti-Chk1, anti-Chk2, anti-Rvb1, and anti-β-actin antibodies are shown. (B) Colocalization of Rvb1 with phospho-H2AX after DNA damage. 293T cells were transfected with a Flag-Rvb1-expressing plasmid. Cells were either left untreated (− UV) or irradiated with UV (+ UV). Immunofluorescence analyses using anti-Flag (red) and anti-phospho-H2AX (green) antibodies were performed. The merged images show the colocalization of Rvb1 and phospho-H2AX in DAPI-stained nuclei (blue). H2AXpS139, H2AX phosphorylated on S139. (C) Endogenous Rvb1 interacts with phospho-H2AX. The results of immunoprecipitation (IP) using either preimmune (PI) or immune (I) antibody against Rvb1 from lysates prepared from HeLa cells after UV irradiation are shown. Western blotting was carried out with anti-phospho-H2AX and anti-H2A antibodies. Ten percent inputs (lane 1) are shown in parallel. +, present. (D) Exogenous Rvb1 interacts with phospho-H2AX. 293T cells were transfected with a Flag-Rvb1-expressing plasmid. Cells were either left untreated (−) or irradiated with UV. Cell lysates were used to immunoprecipitate Flag-Rvb1 and subjected to Western blotting using anti-Flag and anti-phospho-H2AX antibodies. Results obtained using inputs of 10% of wild-type levels (input lanes) are shown in parallel. Input lanes of the immunoblot for H2AXpS139 represent a lighter exposure to see the stimulation of phospho-H2AX signals after UV treatment. IgG, immunoglobulin G; HC, heavy chain; LC, light chain; IB, immunoblot. (E) Increased interaction of Rvb1 with H2AX after DNA damage. 293T cells were transfected with an empty vector or a plasmid expressing FLAG-RVB1. Cells were irradiated and harvested at the indicated times after UV treatment. Lysates were immunoprecipitated with anti-Flag antibody, and immunoprecipitates were subjected to Western blotting for H2AX, phospho-H2AX, and Rvb1. +, present; −, absent.

Sudhakar Jha, et al. Mol Cell Biol. 2008 April;28(8):2690-2700.
7.
FIG. 7.

FIG. 7. From: Human Rvb1/Tip49 Is Required for the Histone Acetyltransferase Activity of Tip60/NuA4 and for the Downregulation of Phosphorylation on H2AX after DNA Damage .

Rvb1 is required for the HAT activity of the Tip60 complex. (A) Rvb1 is required for the HAT activity of the Tip60 complex. 293T cells were transfected with a vector or a plasmid expressing FLAG-TIP60 together with a plasmid expressing an shRNA against RVB1 (shRVB1-A) or a control shRNA (shControl). The Tip60 complex was immunoprecipitated using anti-Flag antibody. Equal amounts of immune complexes were incubated for 0 or 10 min with core histones in the HAT assay. Acetylated histone H4 was detected by immunoblotting with anti-acetyl-lysine and anti-histone H4 antibodies. IgG, immunoglobulin G; AcK, acetyl-lysine; AcH4, acetyl histone H4; +, present. (B) Quantitation of HAT activity using anti-acetyl-lysine as measured in the analysis presented in panel A. Means ± SD of results from four experiments are shown. (C) Recombinant Rvb1 (rRvb1) restores the HAT activity of the Tip60 complex to cells depleted of Rvb1. The Flag-Tip60 complex was immunoprecipitated from cells transfected with the indicated plasmids and assayed for HAT activity as described for panel A. Either recombinant His6-Rvb1 (rRvb1) or nonspecific protein (recombinant glutathione S-transferase [rGST]) was added to the Tip60 complex as indicated. IB, immunoblot. (D) Phospho-H2AX coimmunoprecipitates with acetyl-histone H4. Anti-phospho-H2AX antibody was used for immunoprecipitation (IP) from lysates harvested 60 min post-UV irradiation. Immunoblotting was performed with the indicated antibodies. Ten percent inputs (input lane) are shown in parallel. H2AXpS139, H2AX phosphorylated on S139. (E) The expression of nonacetylable histone H4 increased total phospho-H2AX. 293T cells were transfected with the indicated plasmids, and cells were grown in the presence of puromycin for 48 h. Lysates were immunoblotted with anti-phospho-H2AX, anti-H2AX, anti-Myc (for wild-type and mutant histone H4 [H4WT and H4Mut]), anti-Rvb1, anti-PP2A, and anti-β-actin antibodies. pLPCX, plasmid containing the puromycin resistance gene. (F) Wild-type and nonacetylable histone H4 are incorporated equally into chromatin, but the nonacetylable H4 leads to an increase in the phosphorylation of chromatin-associated H2AX. The fractions were immunoblotted for the indicated proteins. The experiment was carried out as described for panel E. Chr, chromatin; Sol, soluble cellular extract separated from chromatin.

Sudhakar Jha, et al. Mol Cell Biol. 2008 April;28(8):2690-2700.

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