rabbit anti–mouse p150caf-1 (Agro-Bio sas)

Agro-Bio sas rabbit anti–mouse p150caf-1

HP1α and <t>p150CAF-1</t> recruitment after DNA damage induction. (A) Experimental scheme for DNA damage induction in spots. To analyze the behavior of HP1α and p150CAF-1 after DNA lesions induced in a pericentric heterochromatin domain, we selected and microirradiated with a 405 nm laser a single chromocenter (blue circle) in Hoechst presensitized cells. Immediately after, we performed live cell imaging on a confocal microscope and monitored the behavior of GFP-tagged proteins at the targeted chromocenter. (B and C) GFP-mHP1α (B) and GFP-mp150 (C) behavior after 405 nm laser irradiation. In mouse Flp-In 3T3 cells transiently transfected with pcDNA5/FRT+GFP-mHP1α plasmid (B) or Flp-In 3T3+GFP-mp150 cells (C), we irradiated a single chromocenter (white circles) as described in A. We show enlarged images of the damaged areas (orange boxes below) and Hoechst staining of live cells before damage induction and at the end of experiment. The yellow arrows on Hoechst images indicate the chromocenter that was damaged. (D) Experimental scheme for DNA damage induction in lines. We sensitized mouse cells with Hoechst for 5 min at 37°C and subsequently performed local 405 nm laser irradiation in parallel lines (blue lines). At the indicated time after DNA damage induction, we permeabilized cells with CSK + Triton X-100 to remove soluble nuclear components and fixed them for subsequent IF analyses. (E) Transient HP1α accumulation at laser-induced DNA damage sites. We treated 3T3 cells as in D and we performed coimmunostaining with anti-HP1α and anti–mouse p150CAF-1 (mp150) antibodies. We show in the 5 min panel enlarged views of a damaged area (orange boxed region) containing a chromocenter that was partially damaged and the neighboring euchromatic region. (F) Persistence of p150CAF-1 at laser-induced DNA damage sites. We treated Flp-In 3T3+GFP-mp150 cells as previously. We performed immunostaining with an anti-γH2AX antibody and visualized p150CAF-1 by the direct detection of GFP signal. DNA is stained with DAPI. Bars, 10 µm.

Rabbit Anti–Mouse P150caf 1, supplied by Agro-Bio sas, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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HP1α and p150CAF-1 recruitment after DNA damage induction. (A) Experimental scheme for DNA damage induction in spots. To analyze the behavior of HP1α and p150CAF-1 after DNA lesions induced in a pericentric heterochromatin domain, we selected and microirradiated with a 405 nm laser a single chromocenter (blue circle) in Hoechst presensitized cells. Immediately after, we performed live cell imaging on a confocal microscope and monitored the behavior of GFP-tagged proteins at the targeted chromocenter. (B and C) GFP-mHP1α (B) and GFP-mp150 (C) behavior after 405 nm laser irradiation. In mouse Flp-In 3T3 cells transiently transfected with pcDNA5/FRT+GFP-mHP1α plasmid (B) or Flp-In 3T3+GFP-mp150 cells (C), we irradiated a single chromocenter (white circles) as described in A. We show enlarged images of the damaged areas (orange boxes below) and Hoechst staining of live cells before damage induction and at the end of experiment. The yellow arrows on Hoechst images indicate the chromocenter that was damaged. (D) Experimental scheme for DNA damage induction in lines. We sensitized mouse cells with Hoechst for 5 min at 37°C and subsequently performed local 405 nm laser irradiation in parallel lines (blue lines). At the indicated time after DNA damage induction, we permeabilized cells with CSK + Triton X-100 to remove soluble nuclear components and fixed them for subsequent IF analyses. (E) Transient HP1α accumulation at laser-induced DNA damage sites. We treated 3T3 cells as in D and we performed coimmunostaining with anti-HP1α and anti–mouse p150CAF-1 (mp150) antibodies. We show in the 5 min panel enlarged views of a damaged area (orange boxed region) containing a chromocenter that was partially damaged and the neighboring euchromatic region. (F) Persistence of p150CAF-1 at laser-induced DNA damage sites. We treated Flp-In 3T3+GFP-mp150 cells as previously. We performed immunostaining with an anti-γH2AX antibody and visualized p150CAF-1 by the direct detection of GFP signal. DNA is stained with DAPI. Bars, 10 µm.

Journal: The Journal of Cell Biology

Article Title: HP1α recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair

doi: 10.1083/jcb.201101030

Figure Lengend Snippet: HP1α and p150CAF-1 recruitment after DNA damage induction. (A) Experimental scheme for DNA damage induction in spots. To analyze the behavior of HP1α and p150CAF-1 after DNA lesions induced in a pericentric heterochromatin domain, we selected and microirradiated with a 405 nm laser a single chromocenter (blue circle) in Hoechst presensitized cells. Immediately after, we performed live cell imaging on a confocal microscope and monitored the behavior of GFP-tagged proteins at the targeted chromocenter. (B and C) GFP-mHP1α (B) and GFP-mp150 (C) behavior after 405 nm laser irradiation. In mouse Flp-In 3T3 cells transiently transfected with pcDNA5/FRT+GFP-mHP1α plasmid (B) or Flp-In 3T3+GFP-mp150 cells (C), we irradiated a single chromocenter (white circles) as described in A. We show enlarged images of the damaged areas (orange boxes below) and Hoechst staining of live cells before damage induction and at the end of experiment. The yellow arrows on Hoechst images indicate the chromocenter that was damaged. (D) Experimental scheme for DNA damage induction in lines. We sensitized mouse cells with Hoechst for 5 min at 37°C and subsequently performed local 405 nm laser irradiation in parallel lines (blue lines). At the indicated time after DNA damage induction, we permeabilized cells with CSK + Triton X-100 to remove soluble nuclear components and fixed them for subsequent IF analyses. (E) Transient HP1α accumulation at laser-induced DNA damage sites. We treated 3T3 cells as in D and we performed coimmunostaining with anti-HP1α and anti–mouse p150CAF-1 (mp150) antibodies. We show in the 5 min panel enlarged views of a damaged area (orange boxed region) containing a chromocenter that was partially damaged and the neighboring euchromatic region. (F) Persistence of p150CAF-1 at laser-induced DNA damage sites. We treated Flp-In 3T3+GFP-mp150 cells as previously. We performed immunostaining with an anti-γH2AX antibody and visualized p150CAF-1 by the direct detection of GFP signal. DNA is stained with DAPI. Bars, 10 µm.

Article Snippet: Rabbit anti-53BP1 (IB and IF, 1:500; NB 100-304; Novus Biologicals; ); mouse anti–pyrimidine 6,4-pyrimidone photoproduct (6,4-PP; IF, 1:1,000; KTM50; Kamiya Biomedicals; ; ) after 10 min of denaturation in 4 M HCl at RT before blocking; rabbit anti-ATM phospho-Ser1981 (IB and IF, 1:500; 600-401-398; Rockland; ); mouse anti-BRCA1 (IF, 1:500; Ab-1 [EMD] and ab16781 [Abcam]; ); rabbit anti–mouse p60CAF-1 (IF, 1:500; AgroBIO; ); rabbit anti–human p60CAF-1 (IF, 1:250; AgroBIO; ); rabbit anti–mouse p150CAF-1 (IF, 1:1,000; AgroBIO; ); mouse anti–human p150CAF-1 (IB and IF, 1:1,000; ab7655; Abcam; ); mouse anti–cyclobutane pyrimidine dimer (CPD; IF, 1:2,000; KTM53; Kamiya Biomedicals; ; ) after 5 min of denaturation in 0.5 M NaOH at RT before blocking; mouse anti-Chk1 (IB, 1:500; sc-8408; tebu-bio); rabbit anti-Chk1 phospho-Ser317 (IB, 1:1,000; 2344; Cell Signaling Technology; ; ); mouse anti-Chk2 (IB, 1:500; ab3292; Abcam; ); rabbit anti-Chk2 phospho-Thr68 (IB, 1:1,000; 2661; Cell Signaling Technology; ); mouse anti-CtIP (IB, 1:50; provided by R. Baer; ); rabbit anti-FANCD2 (IB, 1:1,000; IF, 1:500; NB 100-182; Novus Biologicals; ; ); rabbit anti-FANCD2 (IB, 1:1,000; IF, 1:500; ab2187; Abcam; ); mouse anti-GFP (IF, 1:500; 11 814 460; Roche; ); rabbit anti-GFP (IF, 1:1,000; 8372; Takara Bio Inc.); mouse anti–H2A-ub (IF, 1:500; 05-678; Millipore; ; ); rat anti-HA (IF, 1:250; 11 867 431 001; Roche); mouse anti-γH2AX (IF- 1:2,000; 05-636; Millipore; ); rabbit anti-γH2AX (1:500; 07-164; Millipore; ; ); rabbit anti-H3K9me3 (IF, 1:500; 07-442; Millipore; ; ); mouse anti-HP1α (IF, 1:1,000; 2HP-1H5-AS; Euromedex; ); rabbit anti-HP1α (IB, 1:1,000; IF, 1:2,000; H-2164, Sigma-Aldrich; ); mouse anti-HP1β (IF, 1:1,000; 1MOD-1A9; Euromedex; ; ); mouse anti-HP1γ (IF, 1:1,000; 2MOD-1G6; Euromedex; ; ); rabbit anti–KAP-1 (IB and IF, 1:500; A300-274; Bethyl Laboratories, Inc.; ); goat anti-Ku80 (IF, 1:500; sc-1484; Santa Cruz Biotechnology, Inc.; ; ); sheep anti-MDC1 (IF, 1:500; GTX 10948; GeneTex; ; ); rabbit anti-NBS1 phospho-Ser343 (IF, 1:500; NB 100-284; Novus Biologicals; ); rabbit anti-p53 phospho-Ser15 (IB, 1:500; 9284; Cell Signaling Technology; ; ); rabbit anti–human RAD51 (IB, 1:1,000; IF, 1:500; PC130; EMD; ); rabbit anti-RPA32 (IB, 1:500; 691-P1ABX; Neomarkers); rabbit anti-RPA32 phospho-Ser4/Ser8 (IB, 1:1,000; A300-245A; Bethyl Laboratories Inc.); rabbit anti-XRCC4 (IF, 1:500; ab2857; Abcam; ); rabbit anti-XRCC4 (IB, 1:1,000; AHP387; Serotec; ); and mouse α-tubulin (IB, 1:20,000; T 9026; Sigma-Aldrich), which was used as loading control.

Techniques: Live Cell Imaging, Microscopy, Irradiation, Transfection, Plasmid Preparation, Staining, Immunostaining

p150CAF-1–dependent corecruitment of HP1α and KAP-1 to laser-induced DNA lesions. (A and B) Confirmation of siRNA-mediated knockdown. We checked by immunostaining (A) and immunoblotting (B) the efficiency of depletion by using antibodies against human p150CAF-1 (hp150), human p60CAF-1 (hp60), HP1α, and KAP-1. We used α-tubulin as a loading control. (C) p150CAF-1-dependent recruitment of HP1α and KAP-1 to laser-induced DNA damage sites. We transfected U2OS cells with control siRNA against GFP (sicont), p150CAF-1 (sip150), or p60CAF-1 (sip60). 72 h later, we treated the cells as in , with CSK + Triton X-100 permeabilization and fixation within 5 min after a local laser irradiation. We performed immunostaining with antibodies against HP1α, KAP-1, and γH2AX. (D) Co-dependence of HP1α and KAP-1 recruitment to laser-induced DNA damage. We treated U2OS cells as previously, except that we used siRNA against HP1α (siHP1α) and KAP-1 (siKAP-1). Then, we performed immunostaining with antibodies against p150CAF-1, HP1α, KAP-1, and γH2AX. DNA was stained with DAPI. Bars, 10 µm.

Journal: The Journal of Cell Biology

Article Title: HP1α recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair

doi: 10.1083/jcb.201101030

Figure Lengend Snippet: p150CAF-1–dependent corecruitment of HP1α and KAP-1 to laser-induced DNA lesions. (A and B) Confirmation of siRNA-mediated knockdown. We checked by immunostaining (A) and immunoblotting (B) the efficiency of depletion by using antibodies against human p150CAF-1 (hp150), human p60CAF-1 (hp60), HP1α, and KAP-1. We used α-tubulin as a loading control. (C) p150CAF-1-dependent recruitment of HP1α and KAP-1 to laser-induced DNA damage sites. We transfected U2OS cells with control siRNA against GFP (sicont), p150CAF-1 (sip150), or p60CAF-1 (sip60). 72 h later, we treated the cells as in , with CSK + Triton X-100 permeabilization and fixation within 5 min after a local laser irradiation. We performed immunostaining with antibodies against HP1α, KAP-1, and γH2AX. (D) Co-dependence of HP1α and KAP-1 recruitment to laser-induced DNA damage. We treated U2OS cells as previously, except that we used siRNA against HP1α (siHP1α) and KAP-1 (siKAP-1). Then, we performed immunostaining with antibodies against p150CAF-1, HP1α, KAP-1, and γH2AX. DNA was stained with DAPI. Bars, 10 µm.

Techniques: Immunostaining, Western Blot, Transfection, Irradiation, Staining

Recruitment of HP1α to laser-induced DNA lesions through its interaction with p150CAF-1. (A) Experimental scheme. We transfected U2OS cells with an siRNA specifically designed against the 3′ UTR of hp150CAF-1 (sip150 3′ UTR). 48 h later, we cotransfected pcDNA5/FRT+HA-mHP1α plasmid with GFP-mp150 wt or mutated in its PxVxL motif (ΔPVVVL and V224D mutants). The following day, we performed CSK + Triton X-100 permeabilization and fixation within 5 min after a local laser microirradiation. (B) Confirmation of siRNA-mediated knockdown. We checked by immunoblotting the efficiency of depletion of the siRNA against the 3′ UTR in comparison with the siRNA used in the rest of the experiments, after 48 h of transfection, using α-tubulin as a loading control. (C) Recruitment of HP1α to local laser-induced DNA damage via p150CAF-1. We treated cells as in A, performed immunostaining with antibodies against HA and γH2AX, and visualized p150CAF-1 by the direct detection of the GFP signal. DNA was stained with DAPI. Bars, 10 µm.

Journal: The Journal of Cell Biology

Article Title: HP1α recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair

doi: 10.1083/jcb.201101030

Figure Lengend Snippet: Recruitment of HP1α to laser-induced DNA lesions through its interaction with p150CAF-1. (A) Experimental scheme. We transfected U2OS cells with an siRNA specifically designed against the 3′ UTR of hp150CAF-1 (sip150 3′ UTR). 48 h later, we cotransfected pcDNA5/FRT+HA-mHP1α plasmid with GFP-mp150 wt or mutated in its PxVxL motif (ΔPVVVL and V224D mutants). The following day, we performed CSK + Triton X-100 permeabilization and fixation within 5 min after a local laser microirradiation. (B) Confirmation of siRNA-mediated knockdown. We checked by immunoblotting the efficiency of depletion of the siRNA against the 3′ UTR in comparison with the siRNA used in the rest of the experiments, after 48 h of transfection, using α-tubulin as a loading control. (C) Recruitment of HP1α to local laser-induced DNA damage via p150CAF-1. We treated cells as in A, performed immunostaining with antibodies against HA and γH2AX, and visualized p150CAF-1 by the direct detection of the GFP signal. DNA was stained with DAPI. Bars, 10 µm.

Techniques: Transfection, Plasmid Preparation, Western Blot, Immunostaining, Staining

53BP1 recruitment to laser-induced DNA damage is impaired by the depletion of p150CAF-1, HP1α, and KAP-1. (A) MDC1 recruitment is not altered upon depletion of p150CAF-1, HP1α, or KAP-1. We transfected U2OS cells with the indicated siRNA and treated the cells as in , and we subsequently performed immunostaining with antibodies against MDC1 and γH2AX. (B) Defective recruitment of 53BP1 to DNA damage sites after depletion of p150CAF-1, HP1α, or KAP-1. We treated U2OS cells as in A and we performed coimmunostaining with antibodies against 53BP1 and γH2AX. DNA was stained with DAPI. Bars, 10 µm.

Journal: The Journal of Cell Biology

Article Title: HP1α recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair

doi: 10.1083/jcb.201101030

Figure Lengend Snippet: 53BP1 recruitment to laser-induced DNA damage is impaired by the depletion of p150CAF-1, HP1α, and KAP-1. (A) MDC1 recruitment is not altered upon depletion of p150CAF-1, HP1α, or KAP-1. We transfected U2OS cells with the indicated siRNA and treated the cells as in , and we subsequently performed immunostaining with antibodies against MDC1 and γH2AX. (B) Defective recruitment of 53BP1 to DNA damage sites after depletion of p150CAF-1, HP1α, or KAP-1. We treated U2OS cells as in A and we performed coimmunostaining with antibodies against 53BP1 and γH2AX. DNA was stained with DAPI. Bars, 10 µm.

Techniques: Transfection, Immunostaining, Staining

Depletion of p150CAF-1 or HP1α leads to defects in the DDR and cell survival. (A) Efficient formation of 53BP1 foci requires p150CAF-1 and HP1α. We transfected U2OS cells with the indicated siRNAs and, 72 h later, irradiated the cells with a dose of 8 Gy. At the indicated time after IR, we permeabilized the cells with CSK + Triton X-100 before fixation and performed coimmunostainings with anti-γH2AX and 53BP1 antibodies. DNA was stained with DAPI. Bars, 10 µm. (B and C) Percentage of cells with 53BP1 or γH2AX foci. Each value shown corresponds to the percentage of cells containing at least five foci of 53BP1 (B) or γH2AX (C) and represents the mean of three independent experiments. (D) IR sensitivity of p150CAF-1– and HP1α-depleted cells. We transfected U2OS cells with the indicated siRNA and analyzed their colony forming capacity after different doses of IR. Each value shown corresponds to the percentage of survival relative to the control and represents the mean of at least two experiments. The error bars represent the standard deviation of the mean. (E) Confirmation of siRNA-mediated depletion. On cells derived from this experiment, we performed immunoblotting to check the efficiency of depletions by using antibodies against hp150, HP1α, FANCD2, and RAD51, and we used α-tubulin as a loading control.

Journal: The Journal of Cell Biology

Article Title: HP1α recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair

doi: 10.1083/jcb.201101030

Figure Lengend Snippet: Depletion of p150CAF-1 or HP1α leads to defects in the DDR and cell survival. (A) Efficient formation of 53BP1 foci requires p150CAF-1 and HP1α. We transfected U2OS cells with the indicated siRNAs and, 72 h later, irradiated the cells with a dose of 8 Gy. At the indicated time after IR, we permeabilized the cells with CSK + Triton X-100 before fixation and performed coimmunostainings with anti-γH2AX and 53BP1 antibodies. DNA was stained with DAPI. Bars, 10 µm. (B and C) Percentage of cells with 53BP1 or γH2AX foci. Each value shown corresponds to the percentage of cells containing at least five foci of 53BP1 (B) or γH2AX (C) and represents the mean of three independent experiments. (D) IR sensitivity of p150CAF-1– and HP1α-depleted cells. We transfected U2OS cells with the indicated siRNA and analyzed their colony forming capacity after different doses of IR. Each value shown corresponds to the percentage of survival relative to the control and represents the mean of at least two experiments. The error bars represent the standard deviation of the mean. (E) Confirmation of siRNA-mediated depletion. On cells derived from this experiment, we performed immunoblotting to check the efficiency of depletions by using antibodies against hp150, HP1α, FANCD2, and RAD51, and we used α-tubulin as a loading control.

Techniques: Transfection, Irradiation, Staining, Standard Deviation, Derivative Assay, Western Blot

Journal: The Journal of Cell Biology

Article Title: HP1α recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair

doi: 10.1083/jcb.201101030

Figure Lengend Snippet: HR-mediated DSB repair is impaired by depletion of p150CAF-1, HP1α, and KAP-1. (A) Defective recruitment of RAD51 after depletion of p150CAF-1, HP1α, or KAP-1. We transfected U2OS cells with the indicated siRNAs and treated the cells as in , with CSK + Triton X-100 permeabilization and fixation within 5 min after laser-induced DNA lesions. We subsequently performed immunostaining with antibodies against RAD51 and γH2AX. DNA was stained with DAPI. Bars, 10 µm. (B) HR-mediated gene conversion is impaired after depletion of p150CAF-1, HP1α, or KAP-1. We analyzed the frequency of HR-mediated DNA repair events in U2OS+DR-GFP cells transfected with the indicated siRNAs. Each value shown corresponds to the percentage of HR relative to the control (sicont) and represents the mean of three independent experiments. The error bars represent the standard deviation of the mean. (C) Cell cycle profile of siRNA-transfected cells. In parallel, we monitor the cell cycle status of the siRNA-treated cells by flow cytometry analysis. (D) Confirmation of siRNA-mediated depletion. We performed immunoblotting to confirm that the indicated siRNA-mediated depletions were efficient in experiments from B. We used RAD51 as loading control. (E) Phosphorylation of the middle subunit of RPA is affected after depletion of HP1α. We transfected U2OS cells with the indicated siRNAs. 48 h later, we treated the cells with 1 µM camptothecin (CPT). At the indicated time points, we harvested the cells and performed immunoblotting with antibodies against RPA32, RPA32 S4/S8, HP1α, and CtIP. We used α-tubulin as loading control. (F) Defective recruitment of BRCA1 after depletion of HP1α. We transfected U2OS cells with the indicated siRNAs and treated the cells as in , with CSK + Triton X-100 permeabilization and fixation within 5 min after laser irradiation. We subsequently performed immunostaining with antibodies against BRCA1 and γH2AX. DNA was stained with DAPI. Bars, 10 µm.

Techniques: Transfection, Immunostaining, Staining, Standard Deviation, Flow Cytometry, Western Blot, Irradiation

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