anti top3α  (Proteintech)

Journal: Nucleic Acids Research

Article Title: Resolution of sequence divergence for repeat-mediated deletions shows a polarity that is mediated by MLH1

doi: 10.1093/nar/gkac1240

Figure Lengend Snippet: Components of mismatch repair and the BLM-TOP3α-RMI1/2 (BTR) complex suppress RMDs, whereas several other factors promote these events. ( A ) Shown are effects of siRNAs targeting 55 factors on frequency of RMDs at both 16 bp and 9.1 kb DSB/repeat distances for 1%RMD-GFP. Frequencies are normalized to transfection efficiency and parallel siCTRL (= 1). Genes are ranked by the fold-effect relative to siCTRL at 9.1 kb. All siRNAs tested n = 2, and those with ≥1.5-fold effect from these trials were tested a total of n = 4. Grey: n = 2, black: n = 4, red: n = 4 and also ≥1.5-fold effect relative to siCTRL. ( B ) Shown are the effects of the siRNAs targeting 55 factors on frequency of RMDs at both 16 bp and 9.1 kb DSB/repeat distances for RMD-GFP. Colors and analysis as in (A). ( C ) Ratio of divergent versus identical RMD frequencies. For the data shown in (A) and (B), shown is the ratio of RMD frequencies from 1% RMD-GFP divided by RMD-GFP. Grey: n = 2, black: n = 4. As above, genes are ranked by the fold-effect relative to siCTRL at 9.1 kb. Data are represented as mean values ± SD.

Article Snippet: Blots were probed with antibodies for CtIP (Active Motif 61141), BLM (Bethyl Laboratories A300-110A), MLH1 (Abcam, ab92312), MSH6 (Proteintech, 18120–1AP), MSH2 (Bethyl Laboratories, A300-452), EXO1 (Bethyl Laboratories, A302-640A), TOP3α (Proteintech, 14525I-AP), FLAG (Sigma, A8592) and ACTIN (Sigma, A2066).

Techniques: Transfection

Journal: Nucleic Acids Research

Article Title: Resolution of sequence divergence for repeat-mediated deletions shows a polarity that is mediated by MLH1

doi: 10.1093/nar/gkac1240

Figure Lengend Snippet: TOP3α suppresses RMDs in a manner that is distinct from MLH1. ( A ) Shown are the frequencies of the RMD events depicted in the diagram in Figure (i.e. six different DSB/repeat distances with RMD-GFP, 1% RMD-GFP and 3% RMD-GFP) for WT mESCs transfected with siCTRL and EV, siTop3a and EV, and siTop3α and TOP3α expression vector. n = 6. * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005, **** P < 0.0001, unpaired t -test with Holm−Sidak correction. ( B ) Immunoblotting analysis of TOP3α and ACTIN in WT mESCs transfected with either siCTRL EV, siTop3α EV or siTop3a with TOP3α expression vector. Endogenous mouse TOP3α was not detected, likely due to the immunogen being human TOP3α. Also shown is immunoblotting analysis of BLM and ACTIN in WT mESCs transfected with either siCTRL, siBlm or siTop3α. ( C ) Shown is the frequency of top strand base retention performed as in Figure , at the 16 bp DSB/repeat distance for WT (siCTRL) and WT siTop3α. WT (siCTRL) values are the same as in Figure . n = 3. * P ≤ 0.05, unpaired t -test. ( D ) Shown is the effect of siRNAs targeting TOP3α (siTop3α) on three RMD events (9.1 kb DSB/repeat distance, RMD-GFP, 1% GFP-GFP, 3% RMD-GFP) in WT, Mlh1 − / − and Msh2 − / − , mESCs. Frequencies are normalized to transfection efficiency and parallel siCTRL (= 1). n = 6. ** P ≤ 0.005, *** P ≤ 0.0005, **** P < 0.0001, unpaired t -test for siCTRL versus siTop3α, and unpaired t -test using Holm−Sidak correction for effect of siTop3α in WT versus the other genetic backgrounds. ( E ) Shown is the analysis as in (D), but using the 16 bp DSB/repeat distance. n = 6. Statistics as in (D), except with ** P ≤ 0.005. Data are represented as mean values ± SD. The † symbol notes that endogenous mouse Top3α is not readily detected by this antibody raised against the human protein.

Article Snippet: Blots were probed with antibodies for CtIP (Active Motif 61141), BLM (Bethyl Laboratories A300-110A), MLH1 (Abcam, ab92312), MSH6 (Proteintech, 18120–1AP), MSH2 (Bethyl Laboratories, A300-452), EXO1 (Bethyl Laboratories, A302-640A), TOP3α (Proteintech, 14525I-AP), FLAG (Sigma, A8592) and ACTIN (Sigma, A2066).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: Molecular cell

Article Title: Topoisomerase 3α Is Required for Decatenation and Segregation of Human mtDNA

doi: 10.1016/j.molcel.2017.11.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-Top3α , Proteintech Group , Cat#14525-1-AP; RRID: AB_2205881.

Techniques: Virus, Isolation, Recombinant, Labeling, Filtration, DNA Library Preparation, Sequencing, Cell Culture, Negative Control, Plasmid Preparation, Software