Image mastertm 2d platinum 6

After staining, we scanned gels using a calibrated UMAX PowerLook 1120 scanner running LabScan software (GE Healthcare, www.gehealthcare.com). We used Image Master^TM 2D Platinum 6.0 software (GE Healthcare, www.gehealthcare.com) to analyze the gels. We analyzed differences between corresponding spots in each set of gels (NES2Y exposed to DMSO and p,p‘-DDT 150 μM). We selected spots with an approximately twofold (or bigger) difference in expression between the cell lysate exposed to DMSO and the cell lysate exposed to 150 μM DDT as spots with a different expression. We determined the statistical significance of changes in protein expression using the student´s t-test. Spots with significantly different intensities were cut and sent for MS analysis.

Cells (approximately 1 × 10⁶ cells per sample) were seeded and after a 24-h pre-incubation period (allowing cells to attach), the culture medium was replaced with a serum-free medium containing 2% BSA with or without FA(s) (SA, a combination of SA and OA, or OA alone) at required concentrations. Cells were placed inside a standard incubation cabinet (20% oxygen level, external normoxia) or inside chambers of a specific incubation cabinet [31 (link)] in which oxygen levels were 4% (moderate hypoxia) or 1% (strong hypoxia). After relevant time of incubation, cells were harvested and the western blot analysis was performed as described previously [4 (link)]. All primary antibodies were used in a 1:1000 dilution. The chemiluminescent signal was detected using a Carestream Gel Logic 4000 PRO Imaging System equipped with Carestream Molecular Imaging Software (Carestream Health, New Haven, CT, USA), which was used for image acquisition. Image Master^TM 2D Platinum 6.0 software (GE Healthcare, Uppsala, Sweden) was used to obtain data for densitometric analysis.

Gels were scanned using a UMAX PowerLook 1120 scanner and Labscan software (GE Healthcare, Uppsala, Sweden) at 600 dpi. Images were analyzed using Image Master^TM 2D Platinum 6.0 software (GE Healthcare, Uppsala, Sweden). Spots were manually detected and analyzed using three independent sets of gels (sensitive MCF7 versus paclitaxel-resistant MCF7/PacR). The statistical significance of the change in expression of matched spots was determined using the Student´s t-test. Spots with at least a two-fold change in the signal were selected for MALDI-TOF mass spectrometry (MS) analysis.