Journal: Nucleic Acids Research

Article Title: The N-terminus of the human RecQL4 helicase is a homeodomain-like DNA interaction motif

doi: 10.1093/nar/gks591

Figure Lengend Snippet: The Sld2-homologous N-terminal domain of RecQL4 interacts directly with TopBP1. ( A ) Schematic depiction of human RecQL4 and TopBP1. ( B ) Specific interaction of the N-terminal domain of RecQL4 fused to the GAL4 DNA-BD with C-terminal part (a.a. 793–1522) of TopBP1 fused to the GAL4-AD in yeast. Interaction was semi-quantitatively assessed by β-galactosidase liquid culture activity assays. β-galactosidase values represent the mean and standard deviations of three independent experiments. ( C ) GST pull-down experiments performed with purified GST, GST–TopBP1 and GST–TopBP1 (a.a. 793–1522) and [ 35 S]–methionine–labelled RecQL4 (a.a. 1–675) (lower panel). Coomassie brilliant blue R250-stained SDS–PAGE gels of the purified GST constructs are presented (upper panel). ( D ) GST pull-down experiments performed with purified GST and GST–RecQL4 (a.a. 1–54) and [ 35 S]-cysteine-labelled full-length TopBP1. Coomassie brilliant blue R250-stained SDS–PAGE gels of the purified GST constructs are presented (upper panel). Input IVT: 10 µl of the in vitro translation product. ( E ) Reciprocal immunoprecipitation of RecQL4 and TopBP1. Cell extracts from human Hek293 cells transiently co-transfected with expression vectors for RecQL4 and ToPBP1 were subjected to immunoprecipitation using antibodies against RecQL4 and TopBP1 as described in ‘Material and Methods’. The precipitates were then analysed by SDS–PAGE and Western blot against the cognate proteins. Controls without antibody or with non-specific rabbit IgG served as negative control and 10% of the input served as an indicator for the (co-)immunoprecipitation efficiency.

Article Snippet: TopBP1 polyclonal antibody (4 μg, Novus Biologicals), and anti-RecQL4 (a rabbit polyclonal antibody raised against recombinant a.a. 1–115 of human RecQL4) or pre-immune serum were incubated with 20 μl (bed volume) of protein G-Sepharose (GE Healthcare) and 1% BSA in lysis buffer overnight at 4°C.

Techniques: Activity Assay, Purification, Staining, SDS Page, Construct, In Vitro, Immunoprecipitation, Transfection, Expressing, Western Blot, Negative Control

Journal: Nucleic Acids Research

Article Title: The N-terminus of the human RecQL4 helicase is a homeodomain-like DNA interaction motif

doi: 10.1093/nar/gks591

Figure Lengend Snippet: Characterization of the TopBP1 interaction with RecQL4. ( A ) Identification of the RecQL4_N54 BD of TopBP1 by GST pull-down. Pull-down experiments were performed as in C–D with GST-fusion constructs as bait for in vitro translated proteins as indicated. Only TopBP1 (a.a. 1233–1522) shows efficient binding to GST-RecQL4_N54. Purified GST and GST-TopBP1 (a.a. 1233–1264) stained with Coomassie brilliant blue R250 are shown on the right. ( B ) Real-time in vitro SPR binding analysis of RecQL4_N54 to anti-GST antibody captured GST-TopBP1 (a.a. 1233–1522). Sensorgrams of 200, 100, 50, 25, 12.5, 6.25 and 3.13 µM RecQL4_N54 binding injected in triplicate (black lines) are shown overlaid with the best fit derived from a 1:1 interaction model (red lines) (left). Fit of the equilibrium data for TopBP1 (a.a. 1233–1522) binding (middle). The purified GST-TopBP1 (a.a. 1233–1593) used for the experiment stained with Coomassie brilliant blue R250 is shown on the right.

Article Snippet: TopBP1 polyclonal antibody (4 μg, Novus Biologicals), and anti-RecQL4 (a rabbit polyclonal antibody raised against recombinant a.a. 1–115 of human RecQL4) or pre-immune serum were incubated with 20 μl (bed volume) of protein G-Sepharose (GE Healthcare) and 1% BSA in lysis buffer overnight at 4°C.

Techniques: Construct, In Vitro, Binding Assay, Purification, Staining, Injection, Derivative Assay

Journal: The EMBO Journal

Article Title: FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress

doi: 10.1038/s44318-024-00323-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-TopBP1 monoclonal antibody , Cell Signaling Technology , Cat # 14342.

Techniques: Recombinant, Sequencing, shRNA, Modification, Transfection, Protease Inhibitor, Cloning, In Situ, Software