Journal: International Journal of Molecular Sciences

Article Title: DNA Damage Induced MutS Homologue hMSH4 Acetylation

doi: 10.3390/ijms141020966

Figure Lengend Snippet: hMSH4 interacts with hMof. ( A ) Recombinant hMof was produced as a glutathione S -transferase-tagged fusion protein and was co-expressed with hMSH4. Soluble cell lysates were used for GST pull-down analysis. Western blot analysis was performed to detect the expression of hMSH4 protein; (B) Negative controls for GST pull-down assay. In the absence of GST-hMof, glutathione-Sepharose 4B beads could not directly pull down hMSH4 even in the presence of GST tag; ( C ) Co-immunoprecipitation analysis of hMSH4 and hMof interaction in human cells. Myc-hMSH4 and Flag-hMof expression in 293T cells was validated by Western blotting. IR treatment was performed 48 h after transfection. The α-Flag antibody was used to perform co-immunoprecipitation analysis, and co-immunoprecipitated hMSH4 was validated by Western blot analysis.

Article Snippet: Specifically, the hMof coding sequence was amplified by the use of primers hMof F-6 Kpn (5′-GGGGTACCCCCGCGATGGCGGCACAGGG AGCT-3′) and hMof R1389 Bam H (5′-CGCGGATCCGGGCCAGGCTGCTCACTTCTTGGA-3′) and cloned into pPuro-Flag [ ].

Techniques: Recombinant, Produced, Western Blot, Expressing, Pull Down Assay, Immunoprecipitation, Transfection

Journal: International Journal of Molecular Sciences

Article Title: DNA Damage Induced MutS Homologue hMSH4 Acetylation

doi: 10.3390/ijms141020966

Figure Lengend Snippet: hMof mediates hMSH4 acetylation in vitro . ( A ) Western blot analysis of hMSH4 and hMof expression in 293T cells. Cell extracts were prepared 48 h after transfection; ( B ) In vitro acetylation analysis (see Materials and Methods for details). Immunoaffinity purified hMSH4 and hMof from IR-treated and control cells were incubated in the in vitro acetylation reaction buffer for 15 min, and samples were analyzed by immunoblotting; ( C ) Western blot analysis of immunoaffinity purified hMof. When the in vitro acetylation assay was performed with hMof alone, there was no detectable lysine acetylation signal within the range of molecular weights similar to that of hMSH4. This blot served as a specificity control for the in vitro acetylation assay.

Article Snippet: Specifically, the hMof coding sequence was amplified by the use of primers hMof F-6 Kpn (5′-GGGGTACCCCCGCGATGGCGGCACAGGG AGCT-3′) and hMof R1389 Bam H (5′-CGCGGATCCGGGCCAGGCTGCTCACTTCTTGGA-3′) and cloned into pPuro-Flag [ ].

Techniques: In Vitro, Western Blot, Expressing, Transfection, Purification, Control, Incubation, Acetylation Assay

Journal: International Journal of Molecular Sciences

Article Title: DNA Damage Induced MutS Homologue hMSH4 Acetylation

doi: 10.3390/ijms141020966

Figure Lengend Snippet: hMof modulates the effect of hMSH4 on NHEJ-mediated DSB repair and cell survival in response to IR. ( A ) Schematic representation of the NHEJ reporter locus. The relative locations of the ATG start codon, the I- Sce I recognition sites, and the CMV promoter ( P CMV ) are indicated; ( B ) Analysis of the effects of hMof and hMSH4 on NHEJ. Expression constructs encoding I- Sce I, hMof sh-2, and hMSH4 were transfected into the NHEJ reporter cell line 293T/#8-1 as indicated. The hMof knockdown construct, hMof sh-2, was found to be able to silence approximately 90% of hMof protein expression (data not shown). Cells were analyzed by FACS at 48 h post-transfection. Average NHEJ activities of three measurements were graphed. Error bars are standard deviation of the mean; ( C ) Depletion of mys-2 protects wild type C. elegans from IR exposure. Graphs show the survival rate of embryos laid by wild type (N2) and him-14 hermaphrodites exposed to 0 or 60 Gy of IR. Data are the average of at least 5 replicates from two radiation exposures (* p < 0.05).

Article Snippet: Specifically, the hMof coding sequence was amplified by the use of primers hMof F-6 Kpn (5′-GGGGTACCCCCGCGATGGCGGCACAGGG AGCT-3′) and hMof R1389 Bam H (5′-CGCGGATCCGGGCCAGGCTGCTCACTTCTTGGA-3′) and cloned into pPuro-Flag [ ].

Techniques: Expressing, Construct, Transfection, Knockdown, Standard Deviation