Gs 800 imaging systems scanner

Immunoblotting was performed to validate the findings of the mass spectrometry-based quantitative proteomic analysis. The proteins extracted from OC and CTR cells were mixed in six pools (3 OC and 3 CTR, same quantity of each sample), separated by SDS-PAGE 10% or 12%, depending on the molecular mass of protein under evaluation) and blotted on PDVF membranes. The blotted membranes were incubated with antibodies against actin (ACT) (Sigma-Aldrich, 1:5000), betahexosaminidase subunit beta (HEXB) (Santa Cruz, 1:1000), mitochondrial superoxide dismutase (SOD2), (Cell Signaling, 1:5000), collagenase 3 (MMP13) (Santa Cruz, 1:1000), cytochrome P450 1B1 (CYP1B1) (Santa Cruz, 1:1000), catalase (CAT) (Abcam, 1:2000) and tubulin (TUB) (Santa Cruz, 1:5000) at 4 °C overnight and then with the appropriate anti-mouse HRP secondary antibody (Sigma-Aldrich, 1:5000) or anti-rabbit HRP secondary antibody (Sigma-Aldrich 1:5000). Immunoreactivity was detected by chemiluminescence using the ECL system (Bio-Rad). Images were acquired using a GS-800 imaging systems scanner (Bio-Rad). A densitometric analysis was performed with Quantity One 4.5.0 software (Bio-Rad) using tubulin bands as normalization factor. The t-test was carried out for the statistical analysis where p ≤ 0.05 was considered significant.