polyclonal antibodies against rad21  (Bethyl)

Journal: PLoS Genetics

Article Title: A cohesin cancer mutation reveals a role for the hinge domain in genome organization and gene expression

doi: 10.1371/journal.pgen.1009435

Figure Lengend Snippet: A RAD21 enrichment measured by ChIP-seq at the HoxA locus in wildtype and R586W cl1 and cl2. B Heatmap representation of RAD21 and CTCF enrichment, with k-means clustering. Rows are ranked in descending order based on WT RAD21 signal (column 1). C Average signal plots depicting RAD21 enrichment at CTCF sites, a merged list of K27ac peaks (enhancers), and a merged list of K4me3-marked TSSs (promoters) in wildtype and R586W cl1 and cl2. D Overlap of called peaks in wildtype and R586W cl1 and cl2. E Distribution of RAD21 peaks across the given genomic sites in wildtype and R586W mESCs. F Distribution of RAD21 peaks common to both R586W clones but not wildtype mESCs. G Differential analysis using Diffbind of RAD21 signal at peaks common to wildtype and both R586W clones. Red, p-adj<0.1. H Distribution across given genomic sites of peaks with increased or decreased signal in R586W mESCs as measured by Diffbind. For analyses shown, data from 2 biological replicates for each of wildtype, R586W cl1, and R586W cl2 were merged. I Representative western blots showing the levels of indicated proteins in soluble (Sol.) and chromatin-bound (CB) fractions. J Amounts of chromatin-bound protein expressed as a fraction of total (chromatin-bound + soluble) signal for WT, R586W cl1, and R586W cl2. n = 3 biological replicates. All differences not significant as measured by two-way ANOVA. Numerical data are presented in .

Article Snippet: For ChIP, polyclonal antibodies against RAD21 (Bethyl Laboratories, A300-080A), H3K4me3 (Abcam, ab8580), H3K27ac (Active Motif, Carlsbad, CA; 39135), H3K27me3 (Abcam, ab6002) were used.

Techniques: ChIP-sequencing, Clone Assay, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: NudCL2 is an Hsp90 cochaperone to regulate sister chromatid cohesion by stabilizing cohesin subunits

doi: 10.1007/s00018-018-2957-y

Figure Lengend Snippet: NudCL2 depletion results in premature sister chromatid separation. HeLa cells transfected with the indicated siRNAs and vectors for 72 h were processed for the following analyses: a immunofluorescence analysis showed that NudCL2-depleted cells exhibited misaligned chromosomes with one CREST dot associated with only one green Hec1 dot at kinetochores. Cells were stained with an anti-Hec1 antibody and human CREST serum. DNA was visualized by DAPI. b – f Chromosome spread assays with Giemsa staining revealed precocious sister chromatid separation in cells depleted of NudCL2. Western blot analysis displayed efficient suppression of NudCL2, Rad21 and Plk1 ( b ). Actin was used as a loading control. The cells were treated with colcemid for 2.5 h and subjected to chromosome spread analysis ( c, d ). Ectopic expression of RNAi-resistant NudCL2 (NudCL2*) effectively reversed the defects in sister chromatid separation induced by NudCL2 depletion ( e , f ). g , h Chromosome spread assay followed by immunofluorescence analysis with the indicated antibodies confirmed the precocious separation of sister chromatids in NudCL2-depleted cells. The cells grown on coverslips were treated with colcemid and subjected to chromosome spread and immunofluorescence analysis. DNA was stained with DAPI. Mitotic cells in chromosome spread experiments were categorized into four groups according to their chromosomal morphology: (1) arm separated, where the arms of sister chromatids were separated but connected at centromeres; (2) sister separated (separated sister chromatids), where sister chromatids were separated along the whole chromosome, but their pairing was maintained; (3) single chromosomes, where sister chromatids were completely separated and scattered; (4) arm closed, where sister chromatids were connected at both centromeres and arms. Mitotic cells with different chromosomal morphologies were calculated. Quantitative data are presented as the mean ± SD (at least three independent experiments). More than 150 cells were measured in each experiment. Bars, 10 μm. Higher magnifications of the boxed regions are displayed

Article Snippet: For Western blot analysis, antibodies against Rad21 (Bethyl, A300-080A), SA2 (Bethyl, A300-581A), Smc1α (Bethyl, A300-055A), Smc3 (Bethyl, A300-060A), Mau2 (Abcam, ab46906), Sgo1 (Santa Cruz, sc-54329), Plk1 (Santa Cruz, sc-53418) and Hsp90 (Abcam, ab59459) were utilized.

Techniques: Transfection, Immunofluorescence, Staining, Western Blot, Control, Expressing

Journal: Cellular and Molecular Life Sciences

Article Title: NudCL2 is an Hsp90 cochaperone to regulate sister chromatid cohesion by stabilizing cohesin subunits

doi: 10.1007/s00018-018-2957-y

Figure Lengend Snippet: NudCL2 interacts with cohesin subunits and Hsp90 in vitro and in vivo. a GST pull-down assays showed that NudCL2 was associated with cohesin subunits and Hsp90 in vitro. Purified GST or GST-NudCL2 protein was incubated with HeLa cell lysates and subjected to immunoblotting. The inputs of GST and GST-NudCL2 were stained with Coomassie brilliant blue. b , c Endogenous NudCL2 bound to four cohesin subunits and Hsp90 in vivo. Total lysates of HeLa cells were immunoprecipitated with the indicated antibodies or IgGs and processed for Western blotting. d – g NudCL2 protein directly interacted with Smc1α and Smc3 but not Rad21 or SA2 in vitro. His-tagged Rad21, SA2, Smc1α and Smc3 were expressed in Sf9 insect cells and purified with nickel-nitrilotriacetic acid beads. GST or GST-NudCL2 protein was incubated with each of the purified cohesin subunits and subjected to Western analysis with the indicated antibodies

Article Snippet: For Western blot analysis, antibodies against Rad21 (Bethyl, A300-080A), SA2 (Bethyl, A300-581A), Smc1α (Bethyl, A300-055A), Smc3 (Bethyl, A300-060A), Mau2 (Abcam, ab46906), Sgo1 (Santa Cruz, sc-54329), Plk1 (Santa Cruz, sc-53418) and Hsp90 (Abcam, ab59459) were utilized.

Techniques: In Vitro, In Vivo, Purification, Incubation, Western Blot, Staining, Immunoprecipitation

Journal: bioRxiv

Article Title: NudCL2 is an Hsp90 cochaperone to regulate sister chromatid cohesion by stabilizing cohesin subunits

doi: 10.1101/358911

Figure Lengend Snippet: HeLa cells transfected with the indicated siRNAs and vectors for 72 h were processed for the following analyses. A Immunofluorescence analysis showed that NudCL2-depleted cells exhibited misaligned chromosomes with one CREST dot associated with only one green Hec1 dot at kinetochores. Cells were stained with an anti-Hec1 antibody and human CREST serum. DNA was visualized by DAPI. B-F Chromosome spread assays with Giemsa staining revealed precocious sister chromatid separation in cells depleted of NudCL2. Western blot analysis displayed efficient suppression of NudCL2, Scc1 and Plk1 (B). Actin was used as a loading control. The cells were treated with colcemid for 2.5 h and subjected to chromosome spread analysis (C and D). Ectopic expression of RNAi-resistant NudCL2 (NudCL2*) effectively reversed the defects in sister chromatid separation induced by NudCL2 depletion (E and F). G, H Chromosome spread assay followed by immunofluorescence analysis with the indicated antibodies confirmed the precocious separation of sister chromatids in NudCL2-depleted cells. The cells grown on coverslips were treated with colcemid and subjected to chromosome spread and immunofluorescence analysis. Data information: Mitotic cells in chromosome spread experiments were categorized into four groups according to their chromosomal morphology: (i) arm separated, where the arms of sister chromatids were separated but connected at centromeres; (ii) sister separated (separated sister chromatids), where sister chromatids were separated along the whole chromosome, but their pairing was maintained; (iii) single chromosomes, where sister chromatids were completely separated and scattered; (iv) arm closed, where sister chromatids were connected at both centromeres and arms. Mitotic cells with different chromosomal morphologies were calculated. DNA was visualized with DAPI. Scale bars, 10 μm. Quantitative data are presented as the mean ± SD (at least three independent experiments). More than 150 cells were measured in each experiment. Bars, 10 μm. Higher magnifications of the boxed regions are displayed.

Article Snippet: For Western blot analysis, antibodies against Scc1 (Bethyl, A300-080A), SA2 (Bethyl, A300-581A), SMC1 (Bethyl, A300-055A), SMC3 (Bethyl, A300-060A), Scc4 (Abcam, ab46906), Sgo1 (Santa Cruz, sc-54329), Plk1 (Santa Cruz, sc-53418) and Hsp90 (Abcam, ab59459) were utilized.

Techniques: Transfection, Immunofluorescence, Staining, Western Blot, Control, Expressing

Journal: bioRxiv

Article Title: NudCL2 is an Hsp90 cochaperone to regulate sister chromatid cohesion by stabilizing cohesin subunits

doi: 10.1101/358911

Figure Lengend Snippet: NudCL2 interacts with cohesin subunits and Hsp90 in vitro and in vivo. A GST pull-down assays showed that NudCL2 associated with cohesin subunits and Hsp90 in vitro. Purified GST or GST-NudCL2 protein was incubated with HeLa cell lysates and subjected to immunoblotting. The inputs of GST and GST-NudCL2 were stained with Coomassie brilliant blue. B, C Endogenous NudCL2 bound to four cohesin subunits and Hsp90 in vivo. Total lysates of HeLa cells were immunoprecipitated with the indicated antibodies or IgGs and processed for Western blotting. D-G NudCL2 protein directly interacted with SMC1 and SMC3 but not Scc1 or SA2 in vitro. His-tagged Scc1, SA2, SMC1 and SMC3 were expressed in Sf9 insect cells and purified with nickel-nitrilotriacetic acid beads. GST or GST-NudCL2 protein was incubated with each of purified cohesin subunits and subjected to Western analysis with the indicated antibodies.

Article Snippet: For Western blot analysis, antibodies against Scc1 (Bethyl, A300-080A), SA2 (Bethyl, A300-581A), SMC1 (Bethyl, A300-055A), SMC3 (Bethyl, A300-060A), Scc4 (Abcam, ab46906), Sgo1 (Santa Cruz, sc-54329), Plk1 (Santa Cruz, sc-53418) and Hsp90 (Abcam, ab59459) were utilized.

Techniques: In Vitro, In Vivo, Purification, Incubation, Western Blot, Staining, Immunoprecipitation

Journal: bioRxiv

Article Title: NudCL2 is an Hsp90 cochaperone to regulate sister chromatid cohesion by stabilizing cohesin subunits

doi: 10.1101/358911

Figure Lengend Snippet: HeLa cells were transfected with control or the indicated siRNAs and subjected to Western analysis. A SMC1 depletion decreased the stability of SMC3, Scc1 and SA2. B Knockdown of SMC3 reduced the protein levels of SMC1, Scc1 and SA2. C Downregulation of Scc1 caused the decrease in the protein level of SA2, but not SMC1 or SMC3. D Depletion of SA2 had no effect on the other cohesin subunits. Actin, a loading control.

Article Snippet: For Western blot analysis, antibodies against Scc1 (Bethyl, A300-080A), SA2 (Bethyl, A300-581A), SMC1 (Bethyl, A300-055A), SMC3 (Bethyl, A300-060A), Scc4 (Abcam, ab46906), Sgo1 (Santa Cruz, sc-54329), Plk1 (Santa Cruz, sc-53418) and Hsp90 (Abcam, ab59459) were utilized.

Techniques: Transfection, Control, Western Blot, Knockdown