Supersignal west fempto maximum sensitivity substrate

WT or set2Δ cells were grown overnight in YPD and diluted to an OD₆₀₀ of 0.2 and grown to an OD₆₀₀ of ~1.0. Upon reaching log phase, cells were washed twice with water and resuspended in SD media. Ten ODs of cells were collected at each time point and protein was isolated via TCA extraction as previously described (Fillingham et al., 2008 (link)). Extracts were then loaded onto 10% SDS-PAGE gels and transferred to PVDF membrane. Membranes were incubated overnight with the following antibodies: Rps6 (Abcam ab40820), ph-S6K (Cell Signaling 2211S), Set2 (in house), and G6PDH (Sigma A9521). Membranes were then washed in TBS-Tween (50 mM Tris, 150 mM NaCl, and 0.5% Tween 20), incubated in secondary antibody (Jackson Labs), and probed with SuperSignal West Fempto Maximum Sensitivity Substrate (ThermoFisher). Two biological replicates were quantified using ImageJ and the changes in ph-Rps6 signal were averaged.