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. 2017 Jul 29;45(17):10115–10131. doi: 10.1093/nar/gkx674

Figure 6.

Figure 6.

The Pcf11 zinc-binding domains do not participate in CF IA assembly. (A) Pull-down assays of recombinant Pcf11–Clp1 and CF IA. The C421S/C424S point mutations and C564S/C567S point mutations were introduced into the ORF of Pcf11 (311–626) or Pcf11 ΔQ20. Pcf11:Clp1 or CF IA was pulled-down via a His-tag at the N-terminus of Pcf11. Proteins were eluted from the beads with imidazole and analysed by SDS-PAGE followed by Coomassie-Blue staining. Equivalent amounts of native wild-type and mutant TAP-purified CF IA were loaded on SDS–12%-polyacrylamide gels and subjected to silver staining (B) or Western blot (C) analyses. Lanes 1: 5 μl of purified nCF IAWT; lanes 2: 20 μl of nCF IApcf11-18, lanes 3: 20 μl of nCF IApcf11-19 were used. Polyclonal antibodies to each individual CF IA subunits were used to probe the Western blots.