Anti g6pdh

Yeast strains of the indicated genotypes (and their wild-type counterparts) were grown in YPD either at permissive or restrictive temperatures. Overnight-saturated cultures were diluted to an OD₆₀₀ of 0.2 and allowed to grow until they reached an OD₆₀₀ of 1. Five OD₆₀₀ equivalents of cells were lysed using a modified TCA extraction method as described (Keogh et al., 2006a (link), 2006b (link)). 10–20 mg of the lysates were separated by SDS-PAGE and variously probed with the following antibodies: anti-FLAG-M2 [for FLAG tagged Spt6] (Sigma-Aldrich, F1804; 1:5000), anti-G6PDH (Sigma-Aldrich, A9521; 1:100,000), anti-histone H3K4me3 (EpiCypher, 13–0004; 1:5000), anti-histone H3K79me3 (Abcam, ab2651, 1:2500) anti-histone H3K36me3 (Abcam, ab9050, ab9050; 1:1000), anti-histone H3 (EpiCypher, 13–0001; 1:50,000), anti-Spt16 (gift from Tim Formosa University of Utah, 1:5000), anti-H3K36me2 (Active Motif, 39255; 1:1000), anti-Set2 (Generated in the Strahl lab, 1:5000), anti-RNAPII-Ser2P (Active Motif, Clone #3E10, 61084; 1:100), anti-H2BK123ub1 (Cell Signaling Technology, 5546; 1:2000), and anti-H2B (Active Motif, 39237; 1:2000). HRP-conjugated anti-rabbit (GE Healthcare, NA934V; 1:10,000) and anti-mouse secondary (GE Healthcare, NA931V; 1:10,000), antibodies were used at 1:1000 and proteins were detected using ECL Prime or enhanced chemiluminescence ECL (Amersham Biosciences).

Briefly, cells either untreated or treated with α factor for different durations (2, 5, 15, 30, 60 and 90 min) were harvested in TCA (5% final concentration), washed with 10 mM NaN₃, collected by centrifugation and the resulting pellets frozen at -80°C. For MAPK inactivation measurements, cells were treated with 3 μM α factor for 30 min, harvested by centrifugation, washed once, resuspended in pheromone-free medium and harvested at the times indicated. Cell extracts were prepared by glass bead lysis in TCA as described before (Hao et al., 2007 (link)). Protein concentration was determined by Dc protein assay (Bio-Rad). Proteins were resolved by 10% SDS-PAGE, transferred to nitrocellulose and detected by immunoblotting with p44/42 MAPK antibodies at 1:500 (9101L, Cell Signalling Technology), Fus3 antibodies at 1:500 (sc-6773, Santa Cruz Biotechnology, inc.) and anti-G6PDH at 1:50,000 (A9521, Sigma-Aldrich). Immunoreactive species were detected by chemiluminescence detection (Thermo Scientific Pierce ECL Plus) of horseradish peroxidase-conjugated antibodies (anti-rabbit, 170-5046 or anti-goat,sc-2768, Santa Cruz) at 1:10,000. Blots were scanned using Typhoon Trio+ (GE healthcare) and band intensity was quantified using Fiji (National Institute of Health).