Sofmaxpro software v 6

Extracted proteins from fresh frozen LUAD samples that harbored Cyanobacteria and also had matching FFPE samples were collected for analysis of microcystin content by ELISA. Additional and independent of LUAD samples, proteins from LUSC samples and negative controls were similarly targeted for analysis of microcystin. The QuantiPlate Kit for Microcystins High Sensitivity from EnviroLogix Inc. (Portland, ME) was used to perform the ELISA. Each sample was prepared from the protein extract, at a dilution factor of 1:25. Microcystin-YR (Sigma-Aldritch, Inc.) was used as a sample positive control, at concentrations of 1 ppb and 0.5 ppb to fall within the range of the microcystin ELISA kit. All dilutions were prepared using 10% (v/v) methanol in DI water. The ELISA was performed according to manufacturer's instructions (EnviroLogix Inc., LLC, Portland, ME). The ELISA assay was read using a SpectraMax M5 microplate reader (Molecular Devices, LLC, San Jose, California) at a wavelength of 450 nm. The results were analyzed using SofMaxPro software v. 6.5.1 (Molecular Devices, LLC, San Jose, California) provided with the microplate reader, using calibration controls as standards.

Total proteins isolated with the AllPrep DNA/RNA/Protein Mini Kit were quantitated using the BCA Protein Assay Kit (Thermo Scientific Pierce, Grand Island, NY, USA). The Human PARP1 DuoSet ELISA kit and the DuoSet Ancillary Reagent kit from R&D Systems (Minneapolis, MN) were used to perform the ELISA assays as per manufacturer’s instructions [27 (link)]. Extracted protein samples were prepared at a 1:20 or 1:50 dilution for the assay. We used PARP-1 antibody (F-2), catalog number sc-8007 from Santa Cruz Biotechnology. The epitope is for amino acids position 764–1014 at the C-terminus of PARP1 human origin. Recombinant human PARP1 standard provided with the ELISA kit was used to create a seven-point standard curve. A reagent blank was included in the ELISA assays, consisting of the reagent diluent provided in the ancillary reagent kit with no protein sample added. Each standard, sample and reagent blank were run in duplicate when performing the ELISA. Results of the ELISA assays were read using a SpectraMax M5 microplate reader (Molecular Devices, LLC, San Jose, California) at a wavelength of 450 nm, and analyzed using SofMaxPro software v. 6.5.1 (Molecular Devices, LLC, San Jose, California) provided with the microplate reader.