Yeast cultures were grown YPAD (1% yeast extract, 2% bacto-peptone, 2% glucose, and 24 mg/l adenine hemisulfate at 30 °C to A600 = 0.2 and treated with either 50 µM MG132 (American Peptide) or an equivalent volume of DMSO (Sigma). At the indicated time points, samples were collected and the absorbance measured at 600 nm.
For plating assays with YU101, carfilzomib and bortezomib, GAC201 and GAC202 were grown overnight in YPD (1% yeast extract, 2% bactopeptone, 2% glucose) and diluted to A600 = 0.3 in YP (1% yeast extract, 2% bacto-peptone). Serial five-fold dilutions were prepared in YP and spotted onto YPD plates supplemented with various proteasome inhibitor drugs at 10 µM (or 20 µM for carfilzomib). The plates were incubated at 30 °C for 2 days. YU101 and carfilzomib were gifts from Proteolix Inc. Bortezomib was a gift from Millenium Pharmaceuticals.
For plating assays with YU101, carfilzomib and bortezomib, GAC201 and GAC202 were grown overnight in YPD (1% yeast extract, 2% bactopeptone, 2% glucose) and diluted to A600 = 0.3 in YP (1% yeast extract, 2% bacto-peptone). Serial five-fold dilutions were prepared in YP and spotted onto YPD plates supplemented with various proteasome inhibitor drugs at 10 µM (or 20 µM for carfilzomib). The plates were incubated at 30 °C for 2 days. YU101 and carfilzomib were gifts from Proteolix Inc. Bortezomib was a gift from Millenium Pharmaceuticals.
