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. Author manuscript; available in PMC: 2019 May 8.

Published in final edited form as: Biochem J. 2019 Jan 15;476(1):115–135. doi: 10.1042/BCJ20180577

Figure 5. — CusS_cp autophosphorylates using a cis mechanism. (A) Cartoons of the expected and experimental results of autophosphorylation studies with homodimers and heterodimers of CusS_cp. The first column identifies wild-type CusS_cp-short homodimers (CP_short), wild-type CusS_cp-long (CP_long), and total of nine different heterodimers numbered 1–9, which are either of wild-type or carrying mutations with disrupted phosphoacceptor His or ATP binding (indicated by a red circle crossed with a line). CusS_cp-short is represented by blue rectangles and CusS_cp-long is represented by gray rectangles. Phosphorylated CusS_cp is shown in red rectangles and labeled with a P. The second and third columns depict the expected phosphorylation pattern for cis and trans phosphorylation, respectively. The last column summarizes the phosphorylation patterns observed in our experiments. (B) Native-PAGE analysis of the homodimers and heterodimers revealed by Coomassie staining. (C) Autophosphorylation analysis by native-PAGE followed by Western blotting using an anti-1-pHis antibody. (D) Autophosphorylation analysis by SDS-PAGE followed by Western blotting using an anti-1-pHis antibody. Labels short/short, short/long, and long/long were used on the native-PAGE analyses to identify the homodimer or heterodimer. For SDS-PAGE, short and long were used to identify each subunit.

Figure 5. — CusS_cp autophosphorylates using a cis mechanism. (A) Cartoons of the expected and experimental results of autophosphorylation studies with homodimers and heterodimers of CusS_cp. The first column identifies wild-type CusS_cp-short homodimers (CP_short), wild-type CusS_cp-long (CP_long), and total of nine different heterodimers numbered 1–9, which are either of wild-type or carrying mutations with disrupted phosphoacceptor His or ATP binding (indicated by a red circle crossed with a line). CusS_cp-short is represented by blue rectangles and CusS_cp-long is represented by gray rectangles. Phosphorylated CusS_cp is shown in red rectangles and labeled with a P. The second and third columns depict the expected phosphorylation pattern for cis and trans phosphorylation, respectively. The last column summarizes the phosphorylation patterns observed in our experiments. (B) Native-PAGE analysis of the homodimers and heterodimers revealed by Coomassie staining. (C) Autophosphorylation analysis by native-PAGE followed by Western blotting using an anti-1-pHis antibody. (D) Autophosphorylation analysis by SDS-PAGE followed by Western blotting using an anti-1-pHis antibody. Labels short/short, short/long, and long/long were used on the native-PAGE analyses to identify the homodimer or heterodimer. For SDS-PAGE, short and long were used to identify each subunit.