α rad51 ab 1  (Novus Biologicals)

Journal: bioRxiv

Article Title: RAD51AP1 mediates RAD51 activity through nucleosome interaction

doi: 10.1101/2020.12.17.421636

Figure Lengend Snippet: A , Schematic of the NCP and the EMSA protocol. B , EMSA of RAD51AP1-His 6 (0.1 and 0.2 μM) with the NCP (0.2 μM) assessed by ethidium bromide (lanes 3-5) first and then by Imperial protein stain (lanes 3-5). The Western blots show the presence of RAD51AP1 entering the gel only when bound to the NCP (lanes 6-7), not alone (lane 8). C , EMSA of His 6 /FLAG-tagged RAD51AP1 (0.4 and 0.8 μM) with the NCP (0.4 μM) visualized by ethidium bromide (lanes 1-4). A second gel was loaded in duplicate, transferred and probed to histone H2A or RAD51AP1. D , EMSA of His 6 /FLAG-tagged RAD51AP1 (0.4 and 0.8 μM) with the NCP (0.4 μM) visualized by ethidium bromide (lanes 1-3). A second gel was loaded in duplicate, transferred and probed to histone H3 or RAD51AP1. E , EMSA of His6/FLAG-tagged RAD51AP1 (0.1 and 0.2 μM) and of FLAG-tagged RAD54 (0.1 and 0.2 μM) with the NCP (0.2 μM) assessed by ethidium bromide (lanes 3-4 and 5-6, respectively). F , RAD51 does not bind to the NCP. EMSA of RAD51 and RAD51AP1 (0.4 and 0.2 μM, respectively) with the NCP (0.2 μM).

Article Snippet: The primary antibodies that were used are: α-RAD51AP1 (NB100-1129; Novus; 1:5,000; and our own α-RAD51AP1 antibody, as previously described in ( )), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:4,000), α-β-Actin (ab6276; Abcam; 1:3,000), α-H3 (ab1791; Abcam; 1:10,000), α-H2A (GTX1129418; GeneTex; 1:1,000); α-FLAG (F3165; Sigma; 1:1,000);α-MBP (PAI-989; ThermoScientific; 1:5,000).

Techniques: Staining, Western Blot

Journal: bioRxiv

Article Title: RAD51AP1 mediates RAD51 activity through nucleosome interaction

doi: 10.1101/2020.12.17.421636

Figure Lengend Snippet: A , Schematic of the duplex capture assay with His 6 /FLAG-tagged RAD51AP1 and either nucleosome-free DNA ( i.e. , 147 bp dsDNA) or the NCP. B , Qualitative analysis of captured DNA (beads) and DNA in supernatant by agarose gel electrophoresis. C , Quantitative analysis: Symbols are the results from independent experiments. Bars are the means from three independent experiments ± 1 SD; *, P < 0.05; **, P < 0.01; two-way ANOVA. D , Schematic of the D-loop reaction with chromatinized pBluescript II SK (-) plasmid DNA. E , Addition of RAD51AP1 (50, 100 and 200 nM; lanes 2-4) or RAD54 (100 and 200 nM; lanes 5-6) promotes the RAD51-mediated Dloop reaction on chromatinized DNA. F , Quantification of the results. Symbols are the results from independent experiments. Bars are the means from 2-4 independent experiments ± 1SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; multiple t -test analysis. G , Agarose gel to show that wild type RAD51AP1 (100 and 200 nM; lanes 2-3) promotes the RAD51-mediated D-loop reaction on chromatinized DNA, but RAD51AP1-K3WA (100 and 200 nM; lanes 4-5) and RAD51AP1-K7WA (100 and 200 nM; lanes 6-7) are unable to do so. H , Western blots of fractionated extracts of the nuclei from HT1080 and U2OS cells without and after exposure to mitomycin C (MMC). The signals for β-Actin and histone H3 serve as loading and fractionation control, respectively. RAD54 is shown for comparison purposes.

Article Snippet: The primary antibodies that were used are: α-RAD51AP1 (NB100-1129; Novus; 1:5,000; and our own α-RAD51AP1 antibody, as previously described in ( )), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:4,000), α-β-Actin (ab6276; Abcam; 1:3,000), α-H3 (ab1791; Abcam; 1:10,000), α-H2A (GTX1129418; GeneTex; 1:1,000); α-FLAG (F3165; Sigma; 1:1,000);α-MBP (PAI-989; ThermoScientific; 1:5,000).

Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, Western Blot, Fractionation, Control, Comparison

Journal: bioRxiv

Article Title: RAD51AP1 mediates RAD51 activity through nucleosome interaction

doi: 10.1101/2020.12.17.421636

Figure Lengend Snippet: A , To initiate ternary complex formation, F3 (dark grey) associates with a nucleosome on the incoming duplex DNA template, while F1 (light grey) binds to the ssDNA of the RAD51-ssDNA nucleoprotein filament. The very C-terminus of RAD51AP1 (purple) engages with RAD51, as previously shown . B , It is also possible that F1 binds to regions of nucleosome-free DNA within the dsDNA target.

Article Snippet: The primary antibodies that were used are: α-RAD51AP1 (NB100-1129; Novus; 1:5,000; and our own α-RAD51AP1 antibody, as previously described in ( )), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:4,000), α-β-Actin (ab6276; Abcam; 1:3,000), α-H3 (ab1791; Abcam; 1:10,000), α-H2A (GTX1129418; GeneTex; 1:1,000); α-FLAG (F3165; Sigma; 1:1,000);α-MBP (PAI-989; ThermoScientific; 1:5,000).

Techniques: