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. Author manuscript; available in PMC: 2016 May 24.
Published in final edited form as: Mol Microbiol. 2016 Feb 28;100(2):345–361. doi: 10.1111/mmi.13321

Figure 5. PccA interacts with SenC in vitro.

Figure 5

(A) Spheroplasts of MT1131 cells expressing SenC or from a ΔsenC strain were incubated with Strep-tag purified wild type PccA or PccA-4C, which contained four engineered cysteine residues close to the metal binding site. When indicated, the cysteine-specific crosslinker BMH was added. Samples were subsequently analyzed under non-reducing PAGE, transferred to PVDF membranes and decorated with antibodies against PccA or SenC. PccA-SenC XL indicates the crosslinking product. (B) Spheroplasts were prepared from MT1131 cells expressing either SenC or SenCΔC, which corresponds to a SenC derivative, in which the two cysteine residues of the Cu-binding motif were replaced by alanine. In addition, spheroplasts of the ΔccoGHIS strain expressing SenC were analyzed. In the ΔccoGHIS strain, the cbb3-Cox assembly factors CcoGHIS are absent. The upper panel was decorated with antibodies against SenC and the lower panel with antibodies against PccA.