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. Author manuscript; available in PMC: 2018 Oct 1.
Published in final edited form as: Biochim Biophys Acta. 2017 Jul 25;1859(10):2058–2067. doi: 10.1016/j.bbamem.2017.07.014

Figure 4.

Figure 4

(A) Ste2p expression levels of various Cys mutants. Total membranes from cells expressing various mutants were prepared as described in Materials and Methods. The membrane preparations were fractionated by SDS-PAGE under non-reducing conditions; the gel was blotted and probed using antibody against the C-terminal FLAG epitope tag to detect the presence of Ste2p at either the monomer or dimer positions, (B) Effect of reducing agent (β-mercaptoethanol) on dimerization. (C) Effect of ligand binding on dimerization. Membranes were incubated with α-factor (1μM final concentration) prior to SDS-PAGE gel under non-reducing conditions and immunoblotting. Molecular mass markers (kDa) are indicated on the left-hand side. Blots were overexposed to be able to see the mutants. Blots with shorter exposure time were used for accurate quantification of Ste2p band intensity (Fig. 5).