FIG 4.

In vitro CrhR degradation using mixtures of wild-type and crhR_TR extracts. The effect of mixing protein extracts from wild-type and crhR_TR cells grown at 20 and 30°C was tested. (A) A mixture of soluble protein, extracted from wild-type (WT) cells grown at 30°C (source of active degradation machinery) and crhR_TR grown at 20°C (source of the potential 27-kDa CrhR_TR protein substrate), was incubated at 30°C, and CrhR_TR (27 kDa) levels were detected by Western analysis in aliquots taken at the indicated times. (B) The reverse of the experiment described in panel A was performed using cell lysates from crhR_TR grown at 30°C (source of the potential 27-kDa CrhR_TR protein substrate) and wild-type cells grown at 20°C (source of detectable native CrhR). CrhR (55 kDa) levels were detected by Western analysis in aliquots taken at the indicated times.