FIGURE 5.

Steady-state abundance of Gem1 WT and mutant proteins expressed in yeast. A, steady-state abundance of WT and mutant Gem1 proteins expressed from the native GEM1 promoter on a low copy (CEN) plasmid in a gem1Δ strain. B, steady-state abundance of WT and mutant Gem1 proteins expressed from the uninduced MET25 promoter on a low copy plasmid in a gem1Δ strain. Whole cell extracts separated by 8% SDS-PAGE were transferred to membrane and immunoblotted with affinity-purified anti-Gem1p polyclonal primary antibody. Protein bands were detected using a fluorescent IRDye 680-conjugated anti-rabbit secondary antibody followed by scanning on an Odyssey imaging system (Li-Cor Biosciences). The minus sign in the far left lane denotes empty vector. Extract loaded/lane in A is twice the amount loaded/lane in B.