Proteins were extracted using RIPA buffer (Sigma) and protease inhibitor cocktail (Sigma), boiled in sample buffer (BioRad), and separated by SDS-PAGE using 6%% stacking gel and 12 % separating gel (cell sample: 50 μg/lane; medium sample: 20 μl/lane).
Medium samples (10 μl each) were mixed with zymogram sample buffer (BioRad, Hercules, CA, USA) and subjected to SDS-PAGE.
When comparing the YPD medium samples and the YPGal medium samples, 698 RDNPs were identified using an FDR cutoff of 5%, which is equivalent to ~ 2.2% of the yeast genome.
The differential gene expression analysis was performed in cell samples collected directly from the IL-2-containing cultures (samples: 6/0, 43/0, j/0, line 5/0 and S9/0) and after 8-hrs incubation of the lymphoblasts in a standard medium without IL-2, following triple thorough washing of the cells in a standard medium (samples: 6/8, 43/8, j/8, line 5/8 and S9/8).
In the case of the medium samples, E2 and T were measured for ovary incubations, but only T was analyzed for the testis incubations (unpublished data indicate that E2 production by testis samples is generally below detection limits).
The level of LDH that is released upon cell death was measured in culture medium samples (20 µl) collected at 24 h, using the LDH colorimetric assay kit (Roche Pharma, Switzerland) following the manufacturer's instructions (30).
Following the addition of fresh medium, samples A2 and B2 demonstrated that a portion of the DCs remained alive, which is in agreement with our hypothesis concerning the toxicity of the supernatant.
High-resolution analysis of the sugar content in the culture medium sampled 2 h after inoculation showed that there was sorbitol consumption in the reference condition; sorbitol and glucose consumption in the low-glucose condition; and glucose but no sorbitol consumption in the high-glucose condition.
Western blotting of M17, PrPC, and PrP231stop lysates and medium sample from PrP231stop cells cultured overnight in serum-free medium with 3F4 shows the expected glycoforms of PrP in PrPC lysates, and undetectable reactivity in M17 and PrP231stop lysates as expected (Fig. 3D, lanes 1 3).
Each treatment was hybridized with the common reference (M. oryzae grown in complete medium) sample for 17 h at 60 ºC in the dark.
