Preparing total protein extracts from yeast cells

Total protein extracts were prepared as described (Kushnirov, 2000 (link); von der Haar, 2007 (link)) with subtle modifications. Briefly, 8 ml of mid-log phase cells were treated with 10% trichloroacetic acid and pelleted by centrifugation. Cell pellets were washed with 10 ml 70% ethanol and 2x with 1 ml water, then re-suspended in 1 ml 0.2 M NaOH and incubated 10 min on ice. Cells were pelleted, resuspended in 160 x OD₆₀₀ μl loading dye (120 mM Tris–HCl pH 6.8, 4% sodium dodecyl sulfate, 0.02% bromophenol blue, 20% glycerol), heated at 95°C for 10 min, and lysates clarified by centrifugation at 16,000 x g. Proteins were separated on 10% tris-glycine SDS-PAGE gels, transferred to 0.45 μm nitrocellulose membranes (Bio-Rad) and probed overnight at 4°C with mouse anti-HA (1:5,000; Sigma-Aldrich, 12CA5), rabbit anti-PSTAIR (1:5,000; Millipore-Sigma, 06–923), rabbit anti-p44/42 MAPK (1:2,500; Cell Signaling Technology, 9102) or rabbit anti-G6PDH (1:5,000; Sigma-Aldrich, A9521). Secondary anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase were from Jackson ImmunoResearch (115–035-003 or 111–035-003) and used at 1:10,000 dilution for 60 min at 4°C. Immunoblots were developed using Clarity Western ECL Substrate (Bio-Rad) and imaged on a ChemiDoc MP multimode imager (Bio-Rad).