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

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

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