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α hrad51 14b4  (Novus Biologicals)


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    Structured Review

    Novus Biologicals α hrad51 14b4
    ( A ) DSB-triggered protein loadings at the proximal and distal regions. The regions, surrounding the site of I- Sce I, used for ChIP analysis were schematically illustrated. Numbers represent the distance from the site of I- Sce I in base pairs. The levels of I- Sce I expression, at different time points post-transfection, were analyzed by Western blotting with a α-HA antibody. Representative image of ChIP analysis of locus −303/−57 was shown, in which GAPDH was used as a positive control. PCR analysis (primer set: F13/IN2R1) of an unrelated region on 6p21.3 was included as an additional ChIP control. Arrows were used to mark the positions of the PCR products. ( B ) DSB-induced hMRE11, <t>hRad51,</t> hMSH5, hMSH4, and c-Abl loadings were analyzed at the proximal and distal loci. Error bars represent standard deviations from the means of triplicate measurements.
    α Hrad51 14b4, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 95/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/%CE%B1+hrad51+14b4/Rad51+Antibody+(14B4)/pmc03762724-41-14-17
    Average 95 stars, based on 99 article reviews
    α hrad51 14b4 - by Bioz Stars, 2026-10
    95/100 stars

    Images

    1) Product Images from "MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants"

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073284

    ( A ) DSB-triggered protein loadings at the proximal and distal regions. The regions, surrounding the site of I- Sce I, used for ChIP analysis were schematically illustrated. Numbers represent the distance from the site of I- Sce I in base pairs. The levels of I- Sce I expression, at different time points post-transfection, were analyzed by Western blotting with a α-HA antibody. Representative image of ChIP analysis of locus −303/−57 was shown, in which GAPDH was used as a positive control. PCR analysis (primer set: F13/IN2R1) of an unrelated region on 6p21.3 was included as an additional ChIP control. Arrows were used to mark the positions of the PCR products. ( B ) DSB-induced hMRE11, hRad51, hMSH5, hMSH4, and c-Abl loadings were analyzed at the proximal and distal loci. Error bars represent standard deviations from the means of triplicate measurements.
    Figure Legend Snippet: ( A ) DSB-triggered protein loadings at the proximal and distal regions. The regions, surrounding the site of I- Sce I, used for ChIP analysis were schematically illustrated. Numbers represent the distance from the site of I- Sce I in base pairs. The levels of I- Sce I expression, at different time points post-transfection, were analyzed by Western blotting with a α-HA antibody. Representative image of ChIP analysis of locus −303/−57 was shown, in which GAPDH was used as a positive control. PCR analysis (primer set: F13/IN2R1) of an unrelated region on 6p21.3 was included as an additional ChIP control. Arrows were used to mark the positions of the PCR products. ( B ) DSB-induced hMRE11, hRad51, hMSH5, hMSH4, and c-Abl loadings were analyzed at the proximal and distal loci. Error bars represent standard deviations from the means of triplicate measurements.

    Techniques Used: Expressing, Transfection, Western Blot, Positive Control, Control

    ( A ) ChIP analysis was performed in conjunction with RNAi-mediated gene silencing to determine the interdependency of DSB-triggered protein loadings at the proximal region. Controls without RNAi treatment were from Fig. 2B – the data is presented again on this graph for the purpose of comparison. ( B ) Knockdown efficiencies of shRNA encoding construct targeting hMRE11, hRad51, hMSH5, or hMSH4. Due to the difficulty in detecting endogenous hMSH4 in 293T cells by Western blotting, the hMSH4 knockdown efficiency was determined by the use of 293T/f45 cells. ( C ) ChIP analysis of the effects of RNAi on DSB-induced protein loadings at a distal region. Levels of protein loading in the absence of RNAi treatment were from Fig. 2B and included for the purpose of comparison. Error bars represent standard deviations from the means of triplicate measurements.
    Figure Legend Snippet: ( A ) ChIP analysis was performed in conjunction with RNAi-mediated gene silencing to determine the interdependency of DSB-triggered protein loadings at the proximal region. Controls without RNAi treatment were from Fig. 2B – the data is presented again on this graph for the purpose of comparison. ( B ) Knockdown efficiencies of shRNA encoding construct targeting hMRE11, hRad51, hMSH5, or hMSH4. Due to the difficulty in detecting endogenous hMSH4 in 293T cells by Western blotting, the hMSH4 knockdown efficiency was determined by the use of 293T/f45 cells. ( C ) ChIP analysis of the effects of RNAi on DSB-induced protein loadings at a distal region. Levels of protein loading in the absence of RNAi treatment were from Fig. 2B and included for the purpose of comparison. Error bars represent standard deviations from the means of triplicate measurements.

    Techniques Used: Comparison, Knockdown, shRNA, Construct, Western Blot

    ( A ) ChIP analysis of the effects of hMSH5 Y742F on DSB-triggered protein loading at both the proximal and distal regions was carried out with 293T reporter cells expressing hMSH5 or hMSH5 Y742F . Briefly, cells were transfected with pcDNA6/Flag-hMSH5 or Flag-hMSH5 Y742F and selected with 10 µg/ml blasticidin. ( B ) Expression of hMSH5 and hMSH5 Y742F in selected clones was validated by Western blot analysis of approximately equal numbers of hMSH5 and hMSH5 Y742F cells. ( C ) The effects of c-Abl kinase inhibition on DSB-induced protein loading at the proximal and distal regions. 293T reporter cells were pretreated with 4 µM imatinib for 48 hrs prior to the induction of DSB by I- Sce I transfection. ChIP analysis was performed to evaluate DSB-induced hRad51, hMSH5, and hMSH4 chromatin association. ( D ) ChIP analysis of GAPDH promoter performed with α-RNAPII or the mouse IgG in the presence or absence of imatinib treatment. Error bars represent standard deviations from the means of triplicate measurements. Asterisks indicate p<0.05 by Student’s t -test.
    Figure Legend Snippet: ( A ) ChIP analysis of the effects of hMSH5 Y742F on DSB-triggered protein loading at both the proximal and distal regions was carried out with 293T reporter cells expressing hMSH5 or hMSH5 Y742F . Briefly, cells were transfected with pcDNA6/Flag-hMSH5 or Flag-hMSH5 Y742F and selected with 10 µg/ml blasticidin. ( B ) Expression of hMSH5 and hMSH5 Y742F in selected clones was validated by Western blot analysis of approximately equal numbers of hMSH5 and hMSH5 Y742F cells. ( C ) The effects of c-Abl kinase inhibition on DSB-induced protein loading at the proximal and distal regions. 293T reporter cells were pretreated with 4 µM imatinib for 48 hrs prior to the induction of DSB by I- Sce I transfection. ChIP analysis was performed to evaluate DSB-induced hRad51, hMSH5, and hMSH4 chromatin association. ( D ) ChIP analysis of GAPDH promoter performed with α-RNAPII or the mouse IgG in the presence or absence of imatinib treatment. Error bars represent standard deviations from the means of triplicate measurements. Asterisks indicate p<0.05 by Student’s t -test.

    Techniques Used: Expressing, Transfection, Clone Assay, Western Blot, Inhibition

    Related Articles

    Expressing:

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants
    Article Snippet: Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.. Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .. Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.

    Transfection:

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants
    Article Snippet: Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.. Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .. Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.

    Western Blot:

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants
    Article Snippet: Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.. Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .. Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.

    Positive Control:

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants
    Article Snippet: Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.. Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .. Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.

    Control:

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants
    Article Snippet: Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.. Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .. Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.

    Comparison:

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants
    Article Snippet: Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.. Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .. Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.

    Knockdown:

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants
    Article Snippet: Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.. Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .. Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.

    shRNA:

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants
    Article Snippet: Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.. Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .. Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.

    Construct:

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants
    Article Snippet: Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.. Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .. Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.

    Clone Assay:

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants
    Article Snippet: Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.. Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .. Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.

    Inhibition:

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants
    Article Snippet: Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.Reporter cells were transiently transfected with the pCMV(I- Sce I)3xNLS construct , , at 5 hours prior to ChIP analysis, which was performed with the EZ-ChIP kit (Millipore, Billerica, MA) according to manufacturer’s instruction.. Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .. Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.Rabbit IgG (Upstate) was used as a negative control, and α-acetyl histone H3 (Upstate) or α-RNAPII (Upstate) were used as positive controls.



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    Novus Biologicals α hrad51 14b4
    ( A ) DSB-triggered protein loadings at the proximal and distal regions. The regions, surrounding the site of I- Sce I, used for ChIP analysis were schematically illustrated. Numbers represent the distance from the site of I- Sce I in base pairs. The levels of I- Sce I expression, at different time points post-transfection, were analyzed by Western blotting with a α-HA antibody. Representative image of ChIP analysis of locus −303/−57 was shown, in which GAPDH was used as a positive control. PCR analysis (primer set: F13/IN2R1) of an unrelated region on 6p21.3 was included as an additional ChIP control. Arrows were used to mark the positions of the PCR products. ( B ) DSB-induced hMRE11, <t>hRad51,</t> hMSH5, hMSH4, and c-Abl loadings were analyzed at the proximal and distal loci. Error bars represent standard deviations from the means of triplicate measurements.
    α Hrad51 14b4, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novus Biologicals α hrad51 14b4 mab
    Analysis of cisplatin-induced hMSH5 association with chromatin and nuclear foci formation . ( A ) 293T/f-hMSH5 and 293T/f-hMSH5 Y742F cells were treated with 20 μM cisplatin, and cross-linked bulk chromatin was immunoprecipitated by an α-acetyl-histone H3 antibody 5 hrs post-treatment. The levels of chromatin-associated hMSH5 and its levels of phosphorylation were analyzed by Western blotting. hMSH5 RNAi was used to knockdown hMSH5. Equal levels of acetyl-histone H3 and histone H4 were present in the immunoprecipitates. ( B ) Analysis of hMSH4 chromatin association following cisplatin treatment. Chromatin was prepared from 293T/f45 cells treated with cisplatin. The levels of chromatin-associated hMSH5 and hMSH4 were analyzed by Western blotting. hMSH5 RNAi was used to knockdown hMSH5. Mouse IgG was used as a negative control. kDa , molecular weight ( Mr ) in thousands. ( C ) Examination of γ-H2AX foci formation 24 hrs post cisplatin exposure. 293T, 293T/f-hMSH5, 293T/f-hMSH5 Y742F , and 293T cells subjected to hMSH5 RNAi were used for this analysis. Cells possessing greater than 15 foci/nucleus were graphically displayed. ( D ) Immunoblotting analysis of the effectiveness of <t>hRad51</t> or hMSH5 knockdown in 293T cells transfected with pmH1P-Bsd/hRad51 sh-1 or pmH1P-Bsd/hMSH5 sh-2. α-Tubulin was used as a loading control. kDa , molecular weight ( Mr ) in thousands. ( E ) Analysis of hMSH5 and hRad51 knockdown on cisplatin-induced hRad51 and hMSH5 nuclear foci formation. Cells were subjected to 10 μM cisplatin for 2 hrs and were analyzed for hMSH5 foci formation 6 hrs post cisplatin removal. Cells that possessed five or more nuclear foci for hMSH5 or hRad51 were scored. Error bars represent standard deviations of the means of three independent measurements. Statistically significant differences between knockdown and control cells were indicated with asterisks (p < 0.05, Student t -test).
    α Hrad51 14b4 Mab, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ( A ) DSB-triggered protein loadings at the proximal and distal regions. The regions, surrounding the site of I- Sce I, used for ChIP analysis were schematically illustrated. Numbers represent the distance from the site of I- Sce I in base pairs. The levels of I- Sce I expression, at different time points post-transfection, were analyzed by Western blotting with a α-HA antibody. Representative image of ChIP analysis of locus −303/−57 was shown, in which GAPDH was used as a positive control. PCR analysis (primer set: F13/IN2R1) of an unrelated region on 6p21.3 was included as an additional ChIP control. Arrows were used to mark the positions of the PCR products. ( B ) DSB-induced hMRE11, hRad51, hMSH5, hMSH4, and c-Abl loadings were analyzed at the proximal and distal loci. Error bars represent standard deviations from the means of triplicate measurements.

    Journal: PLoS ONE

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants

    doi: 10.1371/journal.pone.0073284

    Figure Lengend Snippet: ( A ) DSB-triggered protein loadings at the proximal and distal regions. The regions, surrounding the site of I- Sce I, used for ChIP analysis were schematically illustrated. Numbers represent the distance from the site of I- Sce I in base pairs. The levels of I- Sce I expression, at different time points post-transfection, were analyzed by Western blotting with a α-HA antibody. Representative image of ChIP analysis of locus −303/−57 was shown, in which GAPDH was used as a positive control. PCR analysis (primer set: F13/IN2R1) of an unrelated region on 6p21.3 was included as an additional ChIP control. Arrows were used to mark the positions of the PCR products. ( B ) DSB-induced hMRE11, hRad51, hMSH5, hMSH4, and c-Abl loadings were analyzed at the proximal and distal loci. Error bars represent standard deviations from the means of triplicate measurements.

    Article Snippet: Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .

    Techniques: Expressing, Transfection, Western Blot, Positive Control, Control

    ( A ) ChIP analysis was performed in conjunction with RNAi-mediated gene silencing to determine the interdependency of DSB-triggered protein loadings at the proximal region. Controls without RNAi treatment were from Fig. 2B – the data is presented again on this graph for the purpose of comparison. ( B ) Knockdown efficiencies of shRNA encoding construct targeting hMRE11, hRad51, hMSH5, or hMSH4. Due to the difficulty in detecting endogenous hMSH4 in 293T cells by Western blotting, the hMSH4 knockdown efficiency was determined by the use of 293T/f45 cells. ( C ) ChIP analysis of the effects of RNAi on DSB-induced protein loadings at a distal region. Levels of protein loading in the absence of RNAi treatment were from Fig. 2B and included for the purpose of comparison. Error bars represent standard deviations from the means of triplicate measurements.

    Journal: PLoS ONE

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants

    doi: 10.1371/journal.pone.0073284

    Figure Lengend Snippet: ( A ) ChIP analysis was performed in conjunction with RNAi-mediated gene silencing to determine the interdependency of DSB-triggered protein loadings at the proximal region. Controls without RNAi treatment were from Fig. 2B – the data is presented again on this graph for the purpose of comparison. ( B ) Knockdown efficiencies of shRNA encoding construct targeting hMRE11, hRad51, hMSH5, or hMSH4. Due to the difficulty in detecting endogenous hMSH4 in 293T cells by Western blotting, the hMSH4 knockdown efficiency was determined by the use of 293T/f45 cells. ( C ) ChIP analysis of the effects of RNAi on DSB-induced protein loadings at a distal region. Levels of protein loading in the absence of RNAi treatment were from Fig. 2B and included for the purpose of comparison. Error bars represent standard deviations from the means of triplicate measurements.

    Article Snippet: Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .

    Techniques: Comparison, Knockdown, shRNA, Construct, Western Blot

    ( A ) ChIP analysis of the effects of hMSH5 Y742F on DSB-triggered protein loading at both the proximal and distal regions was carried out with 293T reporter cells expressing hMSH5 or hMSH5 Y742F . Briefly, cells were transfected with pcDNA6/Flag-hMSH5 or Flag-hMSH5 Y742F and selected with 10 µg/ml blasticidin. ( B ) Expression of hMSH5 and hMSH5 Y742F in selected clones was validated by Western blot analysis of approximately equal numbers of hMSH5 and hMSH5 Y742F cells. ( C ) The effects of c-Abl kinase inhibition on DSB-induced protein loading at the proximal and distal regions. 293T reporter cells were pretreated with 4 µM imatinib for 48 hrs prior to the induction of DSB by I- Sce I transfection. ChIP analysis was performed to evaluate DSB-induced hRad51, hMSH5, and hMSH4 chromatin association. ( D ) ChIP analysis of GAPDH promoter performed with α-RNAPII or the mouse IgG in the presence or absence of imatinib treatment. Error bars represent standard deviations from the means of triplicate measurements. Asterisks indicate p<0.05 by Student’s t -test.

    Journal: PLoS ONE

    Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants

    doi: 10.1371/journal.pone.0073284

    Figure Lengend Snippet: ( A ) ChIP analysis of the effects of hMSH5 Y742F on DSB-triggered protein loading at both the proximal and distal regions was carried out with 293T reporter cells expressing hMSH5 or hMSH5 Y742F . Briefly, cells were transfected with pcDNA6/Flag-hMSH5 or Flag-hMSH5 Y742F and selected with 10 µg/ml blasticidin. ( B ) Expression of hMSH5 and hMSH5 Y742F in selected clones was validated by Western blot analysis of approximately equal numbers of hMSH5 and hMSH5 Y742F cells. ( C ) The effects of c-Abl kinase inhibition on DSB-induced protein loading at the proximal and distal regions. 293T reporter cells were pretreated with 4 µM imatinib for 48 hrs prior to the induction of DSB by I- Sce I transfection. ChIP analysis was performed to evaluate DSB-induced hRad51, hMSH5, and hMSH4 chromatin association. ( D ) ChIP analysis of GAPDH promoter performed with α-RNAPII or the mouse IgG in the presence or absence of imatinib treatment. Error bars represent standard deviations from the means of triplicate measurements. Asterisks indicate p<0.05 by Student’s t -test.

    Article Snippet: Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .

    Techniques: Expressing, Transfection, Clone Assay, Western Blot, Inhibition

    Analysis of cisplatin-induced hMSH5 association with chromatin and nuclear foci formation . ( A ) 293T/f-hMSH5 and 293T/f-hMSH5 Y742F cells were treated with 20 μM cisplatin, and cross-linked bulk chromatin was immunoprecipitated by an α-acetyl-histone H3 antibody 5 hrs post-treatment. The levels of chromatin-associated hMSH5 and its levels of phosphorylation were analyzed by Western blotting. hMSH5 RNAi was used to knockdown hMSH5. Equal levels of acetyl-histone H3 and histone H4 were present in the immunoprecipitates. ( B ) Analysis of hMSH4 chromatin association following cisplatin treatment. Chromatin was prepared from 293T/f45 cells treated with cisplatin. The levels of chromatin-associated hMSH5 and hMSH4 were analyzed by Western blotting. hMSH5 RNAi was used to knockdown hMSH5. Mouse IgG was used as a negative control. kDa , molecular weight ( Mr ) in thousands. ( C ) Examination of γ-H2AX foci formation 24 hrs post cisplatin exposure. 293T, 293T/f-hMSH5, 293T/f-hMSH5 Y742F , and 293T cells subjected to hMSH5 RNAi were used for this analysis. Cells possessing greater than 15 foci/nucleus were graphically displayed. ( D ) Immunoblotting analysis of the effectiveness of hRad51 or hMSH5 knockdown in 293T cells transfected with pmH1P-Bsd/hRad51 sh-1 or pmH1P-Bsd/hMSH5 sh-2. α-Tubulin was used as a loading control. kDa , molecular weight ( Mr ) in thousands. ( E ) Analysis of hMSH5 and hRad51 knockdown on cisplatin-induced hRad51 and hMSH5 nuclear foci formation. Cells were subjected to 10 μM cisplatin for 2 hrs and were analyzed for hMSH5 foci formation 6 hrs post cisplatin removal. Cells that possessed five or more nuclear foci for hMSH5 or hRad51 were scored. Error bars represent standard deviations of the means of three independent measurements. Statistically significant differences between knockdown and control cells were indicated with asterisks (p < 0.05, Student t -test).

    Journal: Molecular Cancer

    Article Title: MutS homologue hMSH5: role in cisplatin-induced DNA damage response

    doi: 10.1186/1476-4598-11-10

    Figure Lengend Snippet: Analysis of cisplatin-induced hMSH5 association with chromatin and nuclear foci formation . ( A ) 293T/f-hMSH5 and 293T/f-hMSH5 Y742F cells were treated with 20 μM cisplatin, and cross-linked bulk chromatin was immunoprecipitated by an α-acetyl-histone H3 antibody 5 hrs post-treatment. The levels of chromatin-associated hMSH5 and its levels of phosphorylation were analyzed by Western blotting. hMSH5 RNAi was used to knockdown hMSH5. Equal levels of acetyl-histone H3 and histone H4 were present in the immunoprecipitates. ( B ) Analysis of hMSH4 chromatin association following cisplatin treatment. Chromatin was prepared from 293T/f45 cells treated with cisplatin. The levels of chromatin-associated hMSH5 and hMSH4 were analyzed by Western blotting. hMSH5 RNAi was used to knockdown hMSH5. Mouse IgG was used as a negative control. kDa , molecular weight ( Mr ) in thousands. ( C ) Examination of γ-H2AX foci formation 24 hrs post cisplatin exposure. 293T, 293T/f-hMSH5, 293T/f-hMSH5 Y742F , and 293T cells subjected to hMSH5 RNAi were used for this analysis. Cells possessing greater than 15 foci/nucleus were graphically displayed. ( D ) Immunoblotting analysis of the effectiveness of hRad51 or hMSH5 knockdown in 293T cells transfected with pmH1P-Bsd/hRad51 sh-1 or pmH1P-Bsd/hMSH5 sh-2. α-Tubulin was used as a loading control. kDa , molecular weight ( Mr ) in thousands. ( E ) Analysis of hMSH5 and hRad51 knockdown on cisplatin-induced hRad51 and hMSH5 nuclear foci formation. Cells were subjected to 10 μM cisplatin for 2 hrs and were analyzed for hMSH5 foci formation 6 hrs post cisplatin removal. Cells that possessed five or more nuclear foci for hMSH5 or hRad51 were scored. Error bars represent standard deviations of the means of three independent measurements. Statistically significant differences between knockdown and control cells were indicated with asterisks (p < 0.05, Student t -test).

    Article Snippet: Primary and secondary antibodies used for these experiments included α-hMSH5 [ ], α-hRad51 (Ab-1) pAb (Calbiochem), α-hRad51 (14B4) mAb (Novus Biologicals Inc.), α-γ-H2AX (Upstate), Oregon Green goat α-mouse IgG and Texas Red goat α-rabbit IgG (Invitrogen, Carlsbad, CA).

    Techniques: Immunoprecipitation, Phospho-proteomics, Western Blot, Knockdown, Negative Control, Molecular Weight, Transfection, Control