Hrp conjugated goat anti mouse secondary antibody

Following incubation with rucaparib (0–10 µM) for 30 min, PARP activity was measured in permeabilised cells at 26 °C in the presence of 350 nM NAD+ and 10 mg/mL 12 mer palindromic oligonucleotide to activate the enzyme, as described previously [25 (link),26 (link)]. Following transfer to a nitrocellulose membrane (GE Healthcare Life, Sciences, Amersham, Buckinghamshire, UK) the product, PAR, was measured with 10H anti-PAR monoclonal antibody (Enzo life sciences, Exeter, UK) overnight at 4 °C and HRP-conjugated goat anti-mouse secondary antibody (Dako, Santa Clara, CA, USA) diluted 1:1000 in PBS-Tween (PBS-T) 5%. Clarity Max ECL Western substrate (Bio-Rad, Hercules, CA, USA) was added to the membrane, chemiluminescence imaged using the GBox and Genesys software (Syngene, Cambridge, UK), and quantified with reference to a standard curve of 0–25 pmol purified PAR (Enzo life sciences, Farmingdale, NY, USA) after subtraction of background reactions in the absence of NAD and oligonucleotide. Baseline PAR levels were measured using 50–100-times the cells used for inhibition experiments and with no oligonucleotide or NAD+ present. Purified PAR standard was diluted accordingly to a concentration of 0–25 pmol.

Cells were lysed in RIPA buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Cell Signaling Technology). Cell lysates were loaded on 10% polyacrylamide gel and blotted onto nitrocellulose membrane. Membranes were further blocked with blocking buffer and incubated with pSTAT3(Tyr705) antibody (clone D3A7, Cell Signaling Technology) according to the manufacturer’s instructions. Afterwards, membranes were incubated with a HRP-conjugated goat anti-rabbit secondary antibody (Dako) and developed with UptiLight HRP Blot Chemiluminescent ECL Substrate (Uptima). Blots were further stripped and reincubated with anti-STAT3 antibody (clone 9D8, Abcam) and HRP-conjugated goat anti-mouse secondary antibody (Dako).

FRT cells expressing CFTR were scraped from plates and lysed in RIPA with Halt protease inhibitor cocktail (Thermo Fisher). Protein was quantitated using the BCA assay (Thermo Fisher), samples were mixed with 4× sample buffer, and incubated at 37°C for 10 minutes. Equal amounts of protein (40 μg) were loaded into each lane, resolved by 8% SDS-PAGE, and blotted onto Nitrocellulose membranes. Blocking was with 5% dry milk in PBS plus 0.1% Tween 20 followed by incubation with 10B6.2 mouse anti-CFTR NBD1 primary antibody (1:500; CFTR Folding Consortium) for 2 hours at room temperature, and subsequent goat anti-mouse HRP conjugated secondary antibody (1:5000; Dako) for 1 hour at room temperature. α-tubulin was probed as a loading control (mouse anti-tubuin antibody, Genetex). Labeled proteins were detected using SuperSignal West Femto (CFTR) or Pico (tubulin) ECL kit (Thermo Fisher) and visualized with a Chemidoc (BioRad). CFTR and tubulin bands were quantitated using ImageLab sortware (BioRad). Intensities of CFTR bands were normalized against α-tubulin within the same lanes.

HSECs were grown to confluence in rat tail collagen-coated 96-well plates (Corning CoStar) and then stimulated (see Stimulation of HSECs), before being fixed in 100% methanol and washed with a buffer solution of PBS with 0.1% BSA (Sigma-Aldrich). Cells were then blocked for nonspecific antibody binding in PBS with 2% goat serum diluted for 45 min. Primary antibody (CD151; Abd Serotec; 5 μg/ml) or mouse IgG1 negative control (clone DAK-GO1 DAKO; 5 μg/ml) were added to the appropriate wells and incubated for 60 min at room temperature. This was followed by washing with PBS/BSA solution and then the addition of goat anti-mouse HRP-conjugated secondary antibody (Dako; 0.2 μg/ml) to each well for 45 min. The cells were then washed again with PBS/BSA before developing with an OPD substrate (1,2-phenylenediamine dihydrochloride tablets; Dako) dissolved in distilled water with 2.5 μl of 30% H₂O₂. The reaction was arrested with a stop solution of 0.5 M H₂SO₄ (Sigma-Aldrich) before absorbance was measured on a Synergy HT automated microplate reader (BioTek) at 490 nm. Mean absorbance values were calculated from triplicate wells and corrected for background absorbance by subtracting absorbance values obtained from triplicate wells of an isotype-matched negative control antibody.

GP88 tissue expression was measured by IHC on sections of tissue from FFPE whole tissue blocks using previously validated and described IHC methodology [21 (link), 22 (link)]. Briefly, for each case, individual 5 micron sections on positively charged microscope slides were deparaffinized with xylene and rehydrated through a graded ethanol series. Antigen retrieval was conducted for 25 min in 0.2 M citrate buffer pH 6.0 in a 94 °C water bath. Staining was carried out on a Dako Autostainer. GP88 was detected in tissue sections by incubation with an anti-human GP88 mouse monoclonal antibody, clone 6B3 from A&G Pharmaceutical Inc. (Precision Antibody Division) Columbia, MD, followed by washing, and incubation with HRP-conjugated secondary goat anti-mouse antibody (Dako, Carpinteria, CA). Bound antibody was detected using DAB as chromogen (Dako). Slides were then washed and counter-stained with Mayer’s Hematoxylin, prior to examination and scoring.