*2.5. Determination of Oxidative and Antioxidative Genes by qRT-PCR*

*Nrf2*, *catalase*, glutathione peroxidase (*gpx*) 1 and 2, superoxide dismutase (*sod*) 1 and 2, and NADPH-oxidase subunits p47phox and gp91phox mRNA levels were detected using the Eco real-time PCR System (Illumina, San Diego, CA, USA) and Fast SYBR® Green Master Mix (Applied Biosystems, Waltham, MA, USA). ARN purification was performed with Tri-Reagent (Merk, Kenilworth, NJ, USA) of PECs obtained from BALB/c mice at 20 dpi, SnPP-treated or untreated, as previously described [17]. Standard amplification conditions were 10 min at 95 ◦C, 40 thermal cycles of 15 s at 95 ◦C, 30 s at 60 ◦C, and 30 s at 72 ◦C, with a final extension of 10 min at 72 ◦C. The following primers were used: *nrf2*-F: 5 - CAGCATGTTACGTGATGAGG-3 , *nrf2*-R: 5 -GCTCAGAAAAGGCTCCATCC-3 , *gpx1*-F: 5 - GGGACTACACCGAGATGAACGA-3 , *gpx1*-R: 5 -ACCATTCACTTCGCACTTCTCA-3 , *gpx2*- F: 5 -GAGGAACAACTACCCGGGACTA-3 , *gpx2-R*: 5 -ACCCCCAGGTCGGACATACT-3 , *sod1*-F: 5 -TGGGTTCCACGTCCATCAGTA-3 , *sod1*-R: 5 -ACCGTCCTT TCCAGCAGTCA-3 , *sod2*-F: 5 -ATTAACGCGCAGATCATGCA-3 , *sod2*-R: 5 -TGTCCCCCACCATTGAACTT-3 , *catalase*-F: 5 -GCGTCCAGTGCGCTGTAGA-3 , c*atalase*-R: 5 -TCAGGGTGGACGTCAGTGAA-3 , *p47phox*-F: 5 -GAGGCGGAGGATCCGG-3 , *p47phox*-R: 5 -TCTTCAACAGCAGCGTACGC-3 , *gp91phox*-F: 5 -CCAGTGAAGATGTGTTCAGCT-3 , *gp91phox*-R: 5 -GCACAGCCAGTAGA

AGTAGA-3 , *gapdh*-F: 5 -ATGACATCAAGAAGGTGGTGAAG-3 , *gapdh*-R: 5 -TCCTTGGAGG CCATGTAGG-3 . Results were expressed as the ratio between each gene under study and GAPDH expression. Relative gene expression levels were calculated using the 2−ΔΔCT method and normalized to GAPDH. All reactions were performed with at least five biological replicates.
