Expressing Cdc34 Fusion Proteins in Yeast

Cdc34 fusion proteins were expressed under the control of the constitutive glyceraldehyde 3-phosphate dehydrogenase, GPD, promoter from a CEN plasmid (p416 GPD) with a URA3 selection marker in S. cerevisiae strain BY4741 pdr5Δ (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pdr5::kanMX4). Cells were grown to mid-log phase and lysed by vortexing with glass beads (BioSpec Products). Protein extracts were prepared and analyzed by Western blotting using standard protocols as described^{6 (link)}. Cdc34 protein was detected with a monoclonal anti-HA antibody (1:5,000; Sigma, #H9658) and an Alexa-800-labelled goat anti-mouse secondary antibody (1:20,000; Rockland Immunochemicals, #610-132-121), and Scs2p was detected by an anti-SCS2 polyclonal antibody (1:1,000; gift from J. Brickner, Northwestern University) and an Alexa-680 goat anti-rabbit secondary antibody (1:20,000; Invitrogen, #A21109). Protein amounts were estimated by direct infrared fluorescence imaging (Odyssey LICOR Biosciences). Original images of Western blots can be found in Supplementary dataset 1.