Journal: The Journal of Cell Biology

Article Title: A handcuff model for the cohesin complex

doi: 10.1083/jcb.200801157

Figure Lengend Snippet: Co-IP and WB analysis of cohesin Rad21–Rad21 interaction. Logarithmically growing 293T or HeLa cells were transfected with appropriate Rad21 plasmids or empty vector (EV). Input (10% of IP) and IP samples were resolved by 7% SDS-PAGE and blotted with the indicated anti-bodies. (A) Flag-Rad21 and Myc-Rad21 coimmunoprecipitated. (B) Myc-Rad21 coimmunoprecipitated endogenous Rad21. Bar graphs show the relative level of Myc-Rad21 and endogenous Rad21 in input (left) and co-IP samples (right). Error bars indicate SEM from three observations. (C) Radom primer PCR amplification of DNA from 293T cell lysates and the elutes of immunoprecipitated samples. The template DNA used for PCR was purified from cell lysates after nuclease treatment as shown in lanes 1–4. The cell lysates for IP with Flag mAb (lanes 5 and 6) or Myc pAb (lanes 7 and 8) agarose-conjugated beads were digested with DNase I and RNase A in lanes 5 and 7 but were not digested in lanes 6 and 8. The amounts of DNA template used for PCR were from 125 μg of protein of cell lysates (lanes 1–4) or 1 mg of protein of cell lysates in IP samples (lanes 5–8). There is no DNA in the negative control (lane 9) and 0.25 ng DNA in the positive control (lane 10). (D) Cell lysates were treated with or without DNase I and RNase A before co-IP. (E) Co-IP of Flag-Rad21 and Myc-Rad21 from cells in which Flag-Rad21 and Myc-Rad21 were expressed separately, and lysates were mixed together before IP was performed (**, lanes 6 and 13) or from cells cotransfected with Flag-Rad21 and Myc-Rad21 (lanes 7 and 14). Black lines indicate that intervening lanes have been spliced out.

Article Snippet: Full-length human cDNA encoding Rad21 (KIAA0078) and Smc1 (KIAA0178) were obtained from the Kazusa DNA Research Institute.

Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, SDS Page, Amplification, Immunoprecipitation, Purification, Negative Control, Positive Control

Journal: The Journal of Cell Biology

Article Title: A handcuff model for the cohesin complex

doi: 10.1083/jcb.200801157

Figure Lengend Snippet: Fluorescent protein fragment complementation assay showing the Rad21–Rad21 interaction and the antiparallel orientation. (A) YFP(NT) or YFP(CT) were fused to either the NT or CT end of Rad21. YFP-fused Rad21 constructs were expressed in 293T cells (lanes 3–6), and their interaction with the cohesin complex was examined by IP of the endogenous Smc3 using rabbit anti-Smc3 antisera (lanes 9–12). *, nonspecific band. (B) 293T cells were cotransfected with YFP(NT)- and YFP(CT)-fused Rad21 plasmids (a total of four combinations). YFP fluorescence was examined under a fluorescent microscope 40 h after transfection. (C) YFP fluorescence–positive 293T and HeLa cells transfected with the combination of YFP(NT)-Rad21 and Rad21-YFP(CT) at 400× magnification. (D) Possible antiparallel orientation of Rad21–Rad21 interactions. Only the combination of plasmids in the top panel results in the fluorescence. Bars, 25 μm.

Article Snippet: Full-length human cDNA encoding Rad21 (KIAA0078) and Smc1 (KIAA0178) were obtained from the Kazusa DNA Research Institute.

Techniques: Protein-Fragment Complementation Assay, Construct, Fluorescence, Microscopy, Transfection

Journal: The Journal of Cell Biology

Article Title: A handcuff model for the cohesin complex

doi: 10.1083/jcb.200801157

Figure Lengend Snippet: Rad21–Rad21 interaction determined by yeast two-hybrid assay. (A) Expression of Rad21 wild type (1–631 aa), CT-L (254–631 aa), and CT (451–631 aa) in yeast was analyzed with WBs using Rad21 CT–specific pAb. (B) A LacZ reporter assay was used to probe the Rad21–Rad21 interaction. Yeast cells cotransfected with pC97 and pC86 empty vector is shown as the negative control. (C) pC97 GAL4 BD and pC86 GAL4 AD were used as the positive control. Positive protein–protein interaction is shown in blue in th LacZ reporter assay.

Article Snippet: Full-length human cDNA encoding Rad21 (KIAA0078) and Smc1 (KIAA0178) were obtained from the Kazusa DNA Research Institute.

Techniques: Y2H Assay, Expressing, Reporter Assay, Plasmid Preparation, Negative Control, Positive Control

Journal: The Journal of Cell Biology

Article Title: A handcuff model for the cohesin complex

doi: 10.1083/jcb.200801157

Figure Lengend Snippet: Knockdown of SA1/SA2 disrupts the Rad21–Rad21 interaction and the formation of the two-ring cohesins. (A and B) SA1 and SA2 are knocked down by respective siRNA 24 h after transfection with tagged Rad21 into 293T cells. (A) Myc-Rad21 cannot coimmunoprecipitate endogenous Rad21 when SA2 is knocked down. (B) Myc-Rad21 and Flag-Rad21 cannot reciprocally coimmunoprecipitate each other when SA2 is down-regulated. (C) Sucrose gradient centrifugation to study cohesin–cohesin interaction after SA2 knockdown. 293T cells were transfected with SA2 siRNA or control siRNA, and, 24 h later, the cells were cotransfected with Myc-Rad21 and Flag-Rad21 for 40 h. Cell lysates were prepared and used in sucrose gradient centrifugation. Rad21, Smc3, and SA2 were analyzed using WB. Sedimentation coefficient is shown on the top of the blot. Input, sample before sucrose gradient centrifugation. (D) Dissociation of cohesin from sister chromatids in SA2 knockdown cells. HeLa cells in the mitosis phase were cytospun onto slides, and immunofluorescent microscopy was performed. Rad21 mAb and human CREST antibodies were used to probe Rad21 (red) and centromere (green), respectively. The nuclear material is visualized by DAPI staining (blue). The centromeres of one chromosome shown in the boxes of merge panels are enlarged on the right. (A and C) Black lines indicate that intervening lanes have been spliced out. Bar, 10 μm.

Article Snippet: Full-length human cDNA encoding Rad21 (KIAA0078) and Smc1 (KIAA0178) were obtained from the Kazusa DNA Research Institute.

Techniques: Knockdown, Transfection, Gradient Centrifugation, Control, Sedimentation, Microscopy, Staining

Journal: The Journal of Cell Biology

Article Title: A handcuff model for the cohesin complex

doi: 10.1083/jcb.200801157

Figure Lengend Snippet: Handcuff model of cohesin complex. (A–C) Different configurations of two-ring models. (D) Handcuff model consists of two rings. (E) Establishment of sister chromatid cohesion. For simplicity, only cohesin complexes are shown on the model. Single-ring cohesin complexes are loaded onto the chromosomes at any stage of the cell cycle. During DNA replication at S phase, each of the rings entraps one chromatin. The handcuff is established when the two Rad21 molecules are paired and tethered either by SA1 or SA2 via interaction with the two Rad21 molecules.

Article Snippet: Full-length human cDNA encoding Rad21 (KIAA0078) and Smc1 (KIAA0178) were obtained from the Kazusa DNA Research Institute.

Techniques: