Immunoprecipitation performance and affinity of the PARP1 nanobody.

<p>(A) Immunoprecipitation of endogenous hPARP1 from whole-cell lysates of HEK293T cells with PARP1 nanotrap. Input (I), flow-through (FT) and bound (B) fractions were analyzed by SDS-PAGE followed by Coomassie Blue staining (left) and western blotting with anti-PARP1 antibody (right). (B) Affinity measurement of the PARP1 nanobody with Biacore SPR. The sensorgrams for the nanobody at different concentrations of hPARP1 are indicated.</p

Determination of the PARP family selectivity and species reactivity of the PARP1 nanobody.

<p>(A) Immunoprecipitation of GFP-tagged hPARP1 (141 kDa), hPARP2 (93 kDa), hPARP3 (87 kDa), hPARP9 (123 kDa) and GFP (27 kDa, negative control) with the PARP1 nanotrap from transiently transfected HEK293T cells. RFP-Trap was used as control. Input (I), flow-through (FT) and bound (B) fractions were separated by SDS-PAGE followed by immunoblotting with anti-GFP antibody. (B) Immunoprecipitation of endogenous PARP1 from mouse (MEF) and hamster (BHK) cells with the PARP1 nanotrap. The fractions were analyzed by SDS-PAGE and immunoblotting with anti-PARP1 antibody.</p

Recruitment of endogenous PARP1 to the DNA damage sites as visualized by the PARP1 chromobody in live human cells.

<p>(A) Live-cell imaging of laser-microirradiated (405 nm laser, 100% power, 1 s) human HeLa, human PC3 cells and hamster BHK cells transiently expressing PARP1 chromobody. Time-lapse imaging was carried out at 1 frame per second with the spinning disc microscope acquiring 9 pre-irradiation and 100 post-irradiation frames. Selected time-frames are shown, yellow circles depict the regions of microirradiation (Ø 1 μm), yellow arrow-heads mark the sites before and after irradiation. Scale bar, 5 μm. (B) Live-cell imaging of carbon ion-irradiated HeLa cells (300 ions per point) transiently expressing PARP1 chromobody. The cells were irradiated with accelerated 55 MeV (total energy) carbon ions (LET in water: 310 KeV/μm). At 0 s yellow dots in a cross-shape mark the prospective sites of irradiation. After irradiation images were acquired every ~4 s. Selected time points are shown. Scale bar, 10 μm.</p

On-bead pADPr chain synthesis with the endogenous hPARP1 immunoprecipitated with the PARP1 nanotrap.

<p>Gel electrophoresis and silver staining of pADPr fractions from <i>in vitro</i> synthesis. Lanes 1–7: commercially available pADPr chains (lane 1, control); reaction with the purified recombinant hPARP1 with NAD+ (lane 2) or without NAD+ (lane 3); on-bead reaction with PARP1 nanotrap-precipitated endogenous hPARP1 with NAD+ (lane 4) or without NAD+ (lane 5); on-bead reaction with GFP-Trap with NAD+ (lane 6) or without NAD+ (lane 7).</p

PARP1 chromobody enables visualization of hPARP1 in human HeLa cells.

<p>(A) PARP1 chromobody fused to TagRFP (red) co-localizes with GFP-PARP1 (green) in nucleoli and nucleoplasm. (B-C) PARP1 chromobody fused to TagGFP (B, green) or fused to TagRFP (C, red) visualizes endogenous hPARP1 in nucleoli and nucleoplasm. Cells were fixed, stained with DAPI (blue) and subjected to epifluorescence imaging. Scale bar, 10 μm. (D-F) Live-cell imaging with the PARP1 chromobody upon compound treatment. HeLa cells transiently expressing either PARP1 chromobody, TagRFP-hPARP1 or TagRFP alone were treated for 2 h with 10 μM camptothecin (D), 0.01 μM actinomycin D (E), or 0.01 μM 4-NQO (F). After subjecting cells to incubation with the compounds, cells were washed and allowed to recover for another 2 h. Time-lapse epifluorescence imaging was carried out in an automated fashion every 15 min during treatments and during recovery. The panels show selected frames of the cells before treatments, after 2 h of treatment and after 2 h of recovery. Scale bar, 5 μm.</p

Endogenous hPARP1 co-precipitates together with the intracellularly expressed PARP1 chromobody.

<p>PARP1 chromobody fused to TagRFP was precipitated using the RFP-affinity resin (RFP-Trap) from a whole-cell lysate of HeLa cells stably expressing the chromobody. TagRFP-transfected HeLa cells served as negative control for non-specific binding. Input (I), flow-through (FT) and bound (B) fractions were subjected to SDS-PAGE and immunoblotting with anti-PARP1 antibody, followed by anti-TagRFP antibody and anti-GAPDH antibody as loading control.</p

Epitope mapping of the PARP1 nanobody by immunoprecipitation of hPARP1 domains.

<p>(A) Schematics depicts hPARP1 domain structure: DNA-binding domain (45 kDa), automodification domain (19 kDa) and catalytic domain (58 kDa). Purified recombinant hPARP1 domains were subjected to immunoprecipitation with the PARP1 nanotrap Input (I), flow through (FT) and bound (B) fractions were analyzed by SDS-PAGE followed by Coomassie Blue staining. (B) GFP- or mCherry-tagged hPARP1 domains were transiently expressed in HEK293T cells: full DNA-binding domain (DBD), DBD constituting zinc fingers (ZnF1, ZnF2, ZnF3), as well as the WGR domain (part of the catalytic domain). The cells were lysed and immunoprecipitated with the PARP1 nanotrap and RFP-Trap or GFP-Trap as control. The fractions were subjected to SDS-PAGE followed by immunoblotting with anti-GFP or anti-RFP antibody.</p

Intracellular F2H analysis of the PARP1 chromobody.

<p>BHK-F2H cells were pairwise co-transfected with the PARP1 chromobody fused to TagRFP and one of the GFP-tagged bait constructs: GFP alone, GFP-hPARP1, GFP-hPARP2, GFP-hPARP3, GFP-hPARP9, wild-type ZnF2-GFP and ZnF2mut-GFP. The cells were fixed, stained with DAPI and subjected to fluorescence microscopy. Upper row, green channel: GFP-fusion proteins are enriched at the “spot” in the nuclei of transfected BHK-F2H cells (arrows). Middle row, red channel: binding of the PARP1 chromobody to the full-length GFP-hPARP1 and to the wild-type ZnF2-GFP is visible as local enrichments of the red fluorescent signals (arrows). Neither interaction of the PARP1 chromobody with hPARP2, 3, or 9, nor interaction with the mutant ZnF2mut-GFP construct (G161T, A188S and T189A) can be observed. Co-transfection with GFP (first column) served as negative control to exclude non-specific binding of the PARP1 chromobody to GFP. Scale bar, 5 μm.</p