Alphaease fc standalone software

Total RNA from exponential phase PA and oxyR mutant bacteria was purified using a RiboPure-Bacteria kit as specified by the manufacturer (Ambion). After treatment with DNase I, 1 μg of total RNA was converted to cDNA by using random primers and reverse transcriptase (Promega). Then, 1 μl of cDNA from either PA PAO1 or oxyR mutant bacteria was used as a template to amplify the genes of interest that were not of the classic antioxidant variety (e.g., catalase/peroxidase) as well as omlA, the latter of which is a constitutively expressed gene and is used frequently as a reliable, internal control [14 (link)] using Taq DNA polymerase. After designated cycles of the RT-PCR were completed, amplified products was analyzed by electrophoresis on 1.2% agarose gels after sampling every 5 cycles between 15–45 cycles at 55°C. Band intensities were determined by using AlphaEase FC StandAlone software (Alpha Innotech). Then, the optimum PCR cycles were used to amplify a 253-bp phnW fragment using both PA PAO1 and oxyR mutant bacteria, respectively.

To determine the expression of CYP1A1 and CYP1B1 enzymes, equal amount of protein (20 μg) obtained from vehicle- and CSC-treated macrophages was loaded on a 10% polyacrylamide gel and electrophoresed, followed by transfer to a PVDF membrane. The membrane was blocked in 5% non-fat dry milk and incubated with primary antibody (1:500–1000 dilution) overnight. Next day, the membrane was washed and incubated with secondary antibody (1:2000 dilution). After washing, the blot was visualized by Luminata crescendo western HRP substrate using the Alpha Innotech FluorChem HD2 gel documentation system (Alpha Innotech, San Leandro, CA), and the densitometric data was analyzed using AlphaEase FC StandAlone software (version 6.0.0.14; Alpha Innotech). Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) was used as an internal loading control to normalize the expression of CYP1A1 and CYP1B1 proteins.