Anti rabbit immunoglobulins hrp

Since only one polyclonal antibody (pAb-V65) was specific for the peptide, we developed a competitive ELISA for detection of V65 peptides. Briefly, 96-well plate was coated with 100μl/well of synthetic V65 peptide (Eurogentec, UK) at 0.5μg/ml and incubated overnight at 4C°. Wells were washed and blocked for 1 hour at RT. Wells were then washed again prior to the addition of standards, consisting of premixed concentrations of synthetic V65 peptide from 0–4μg/ml with polyclonal anti-V65 antibody for 30 minutes in assay buffer. 100μl of the antibody-antigen complexes was then put in duplicate wells and incubated for 1h at RT. Wells were then washed 3 times with washing buffer prior to addition of 100μl of anti-rabbit immunoglobulins-HRP (Dako, UK) conjugate (diluted 1 in 1000) to detect the polyclonal antibody. Assay was developed using OPD substrate. Colour development was stopped with 1N HCL solution and the plate read at 490 nm using a Tecan F50 Absorbance reader (Labtech, UK). For competitive ELISA, the higher the concentration of V65 in the sample, the weaker the optical density (OD) values. A standard curve was generated by nonlinear regression using Graph Pad Prism version 6.05 statistical software.

Whole hearts were homogenized in RIPA lysis buffer and the extract spun at 13200 g, 10 min, 4 °C. Total cell lysates were electrophoresed and transferred to PVDF membrane and blocked with Odyssey Blocking Buffer (Li-Cor Biosciences). The membrane was incubated with primary antibodies for 2 h at room temperature or overnight at 4˚C, washed in TBST washing solution, and incubated in Odyssey IRDye secondary antibodies (1:5000) for 1 h before visualization with the Odyssey Infrared Imaging System (Li-Cor Biosciences). The primary antibodies used: for detection of LaminA/C (Rabbit, 1:500, Cell Signaling) that is specific to an epitope in the first 50 amino acids in LMNA, mSun1 (mouse mAB, clone 12.11, neat, from B. Burke), GFP (mouse, 1:500, Roche), LaminB1 (rabbit mAB, 1:500, Abcam), anti-HA epitope (rat mAB, 3F10, 1:1000, Roche), GAPDH (rabbit, 1:500, Abcam), and control β-tubulin (mouse, Tub 2.1, 1:1000, Sigma). For detection of the AAV9-DNhSun1 transgene, a mouse mAB specific to the C-terminus of human Sun1 (hSun1, clone 9.1, neat, from B. Burke) was used in combination with protein A conjugated to HRP (1:500, Cell Signaling). GAPDH (rabbit, 1:500, Abcam) was used with anti-rabbit Immunoglobulins/HRP (DAKO, 1:2500) as a loading control. Uncropped and unprocessed scans of blots are in the Source Data file.

Whole-cell lysates were prepared from KATOIII and NUGC-4 cells incubated with 0, 25 or 50 nM panobinostat for 24 or 48 h. One hundred or 40 µg of total protein was applied to each lane for the KATOIII or NUGC-4 assays, respectively. The transferred membrane was reacted with a recombinant rabbit anti-ACE2 monoclonal antibody (#ab108252, Abcam), followed by treatment with anti-rabbit immunoglobulins/HRP (#P0448, DakoCytomation) and Amersham ECL Prime Western Blotting Detection Reagent (#RPM2232, GE Healthcare). Then, densitometry measurements were conducted with a LAS-3000 and MultiGauge v3.0 (FujiFilm, Tokyo, Japan). Subsequently, the same membrane was rinsed, processed with blocking buffer, reacted with mouse anti-β-actin monoclonal antibody (#017-24551, Wako) and anti-mouse IgG (H + L)-HRP conjugate (#172-1011, BioRad). Detection and densitometry measurements were performed again as described above.

PARP1 trapping assay was adapted from (Murai et al, 2012 (link)). In brief, 24 h prior the assay, KB2P and KB2P PARG ko cells were seeded on 10-cm dishes to achieve ~90% confluency. Cells were treated with 500 nM olaparib or 10 µM LNT1 or 1 µM PDDX-004 for 2 h, with the last 30 min in the presence of 0.01% MMS, as indicated. After the treatments cells were trypsinized and subsequently lysed to isolate chromatin-bound fractions. Fractionation was performed with Subcellular Protein Fractionation Kit from Thermo Scientific (#78840, Rockford, IL, USA), following the manufacturer’s instructions. Immunoblotting was carried out as described in the previous section (PAR Immunoblotting), using the rabbit polyclonal anti-PARP1 (#9542, Cell Signaling) primary antibody in dilution 1:1000 and the secondary goat polyclonal anti-rabbit immunoglobulins/HRP (Dako), diluted 1:5000.