Western Blot Analysis of Yeast Transcription Factors

Cells were lysed with lysis buffer [1.85 M NaOH, 7.5% 2-Mercaptoethanol]. Proteins were precipitated with 50% Trichloroacetic acid and resuspended in Urea buffer [40 mM Tris pH8, 8.0 M Urea, 5% SDS, 1% 2-Mercaptoethanol]. Cdr1, Pdr1, and Upc2A rabbit polyclonal antibodies were previously described [18 (link),31 (link)]. Mouse anti-HA monoclonal antibody was purchased from Invitrogen. Secondary antibodies were purchased from LI-COR Biosciences. Imaging was performed with Odyssey CLx Imaging System (LI-COR Biosciences) and analyzed by Image Studio Lite Software (LI-COR Biosciences). Detected target band fluorescence intensity was normalized against tubulin fluorescence intensity and compiled from 2 biological replicate experiments and 2 technical replicates in each experiment, giving 4 replicates in total.