FIG. 4.

Detection of disulfide-cross-linked dimers of the mutant Tcp proteins in nonreducing SDS-PAGE followed by immunoblotting. (A) Detection of cross-linked dimers of Tcp. Ice-cold 5% trichloroacetic acid was added to HCB339 (ΔMCP) cells expressing wild-type (WT) or mutant Tcp proteins pretreated with or without 10 mM N-ethylmalemide (NEM). NEM was added to prevent disulfide formation during sample preparation. The NEM-pretreated or untreated samples were collected by centrifugation and were dissolved in nonreducing SDS loading buffer supplemented with 10 mM NEM (lanes labeled with NEM) or 10% 2-mercaptoethanol (lanes labeled with 2ME), respectively. These samples were subjected to nonreducing SDS-PAGE followed by immunoblotting. (B) Methylation patterns of cross-linked and uncross-linked Tcp proteins. HCB339 (ΔMCP) cells expressing wild-type or mutant Tcp were incubated with 15% glycerol (Glyc), distilled water (None), 10 mM citrate (Cit-10), or 50 mM citrate (Cit-50) at 25°C for 30 min. After stimulation, the samples were treated with 10 mM NEM and 5% trichloroacetic acid and were further treated as described above.