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. Author manuscript; available in PMC: 2012 Aug 16.

Published in final edited form as: Biochemistry. 2011 Jul 12;50(32):6841–6854. doi: 10.1021/bi200254h

Effects α-factor antagonist on dimerization of TM5 and TM6 Cys mutants. A: membranes from cells expressing Ste2p cysteine mutants at cytoplasmic ends of the helices and B: membranes from cells expressing Ste2p cysteine mutants in helices that are suggested to be buried in the membrane were treated were incubated with CuP in the presence or absence of α-factor or α-factor antagonist as described in the Methods. The membrane extracts were run on SDS-PAGE gels, The blots were probed with anti-FLAG antibody to detect the presence of Ste2p at either the monomer (M, 53–55 kDa) or dimer (D, 106–110 kDa) positions, respectively. Each preparation was untreated (lanes −, −) or treated with Cu-P (lanes −, +) or with α-factor and Cu-P (lanes α, +) or with α-factor antagonist and Cu-P (lanes a, +).

Effects α-factor antagonist on dimerization of TM5 and TM6 Cys mutants. A: membranes from cells expressing Ste2p cysteine mutants at cytoplasmic ends of the helices and B: membranes from cells expressing Ste2p cysteine mutants in helices that are suggested to be buried in the membrane were treated were incubated with CuP in the presence or absence of α-factor or α-factor antagonist as described in the Methods. The membrane extracts were run on SDS-PAGE gels, The blots were probed with anti-FLAG antibody to detect the presence of Ste2p at either the monomer (M, 53–55 kDa) or dimer (D, 106–110 kDa) positions, respectively. Each preparation was untreated (lanes −, −) or treated with Cu-P (lanes −, +) or with α-factor and Cu-P (lanes α, +) or with α-factor antagonist and Cu-P (lanes a, +).