rabbit antibody against vsvg  (Covance)

Journal: Nucleic Acids Research

Article Title: Conservation of transcriptional regulation by BRCA1 and BARD1 in Caenorhabditis elegans

doi: 10.1093/nar/gkac877

Figure Lengend Snippet: H2A ubiquitylation is a conserved feature of CeBCBD, but does not primarily occur at the predicted location. ( A ) Amino acid sequence alignment of worm (Ce) and human (Hs) histone H2A tails. The residue highlighted in black, K125, is the lysine closest to the three residues targeted by HsBCBD shown in bold. Dashes indicate gaps in the sequence. ( B ) In vitro activity of CeBCBD and HsBCBD toward nucleosomes containing H2A with the worm C-terminal tail sequence (WT) or K125H mutation is demonstrated via western blot for H2A. ( C ) Mean unmodified H2A remaining after 10 and 30 min from two biological and two technical replicates of in vitro H2A ubiquitination assays. Error bars represent the standard deviation. The intensity of unmodified H2A bands in western blots such as (B) were quantified using ImageJ. Means were compared using a Welch's t -test for data with unequal variance (HsBCBD at 30 min) or an unpaired two-sample t-test was used for data with equal variances (all other time points). Statistical significance was determined using a P -value <0.05. An uncropped blot image is available in the supplementary information.

Article Snippet: Blotting was carried out using a 1:10 000 dilution of a rabbit antibody against the VSVG-tag on histone H2A (Millipore Sigma Corp.).

Techniques: Sequencing, Residue, In Vitro, Activity Assay, Mutagenesis, Western Blot, Ubiquitin Proteomics, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Conservation of transcriptional regulation by BRCA1 and BARD1 in Caenorhabditis elegans

doi: 10.1093/nar/gkac877

Figure Lengend Snippet: BRC-1 utilizes a conserved nucleosome binding interface. ( A ) Sequence alignments of the relevant regions of the RING domains and solution structure (PDB 1JM7) of HsBCBD highlighting the sidechains of residues from BRCA1 and BARD1 that are important for binding to the nucleosome in orange and red, respectively. The sidechain of BARD1 R99 that non-covalently engages ubiquitin in E2∼Ub is shown in blue. Zinc coordinating residues that were mutated in families with a history of breast cancer are shown in gray. Western blots comparing the ubiquitylation activity of mutant BRC-1 ( B ) and mutant BRD-1 ( D ) constructs to wild-type CeBCBD (WT). The substrate observed is H2A incorporated in nucleosomes. Means and standard deviations of unmodified H2A at 30 min are presented for BRC-1 mutants ( C ) and BRD-1 mutants ( E ). Asterisk denotes mutants with significant decreases in activity according to Student's t -test with P -value < 0.05, while ‘ns’ denotes activity difference is not significant ( P -value > 0.05). ( F ) BRD-1 homology model generated by SWISS-MODEL (light blue) highlighting the K54 and R55 basic patch in green . The additional loop in BRD-1 that contains these residues is hypothesized to be positioned below the nucleosome binding residues for BARD1 (red). Uncropped blot images are available in the supplementary information.

Article Snippet: Blotting was carried out using a 1:10 000 dilution of a rabbit antibody against the VSVG-tag on histone H2A (Millipore Sigma Corp.).

Techniques: Binding Assay, Sequencing, Ubiquitin Proteomics, Western Blot, Activity Assay, Mutagenesis, Construct, Generated