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. Author manuscript; available in PMC: 2015 Feb 6.
Published in final edited form as: J Mol Biol. 2014 Feb 6;426(3):570–585. doi: 10.1016/j.jmb.2013.10.025

Fig 4.

Fig 4

HoloCcmE must be released from CcmABCD to interact with CcmF. (A) Coomassie blue staining of TALON-purified proteins from ΔGH and ΔGH delAB backgrounds showing purified 54-kDa full-length CcmF. (B) Anti-CcmF immunoblot of purified CcmF proteins showing 54-kDa full-length CcmF. (C) Heme staining of purified CcmF proteins showing free heme (CcmF b-heme) and co-purified 20-kDa holoCcmE. (D) Anti-CcmE immunoblot of purified CcmF proteins showing co-purified 20-kDa CcmE. For (A)-(D), 5 ug purified protein was analyzed. (E) Heme staining of DDM-solubilized membrane fractions from ΔGH and ΔGH delAB backgrounds showing 20 kDa holoCcmE. (F) Anti-CcmE immunoblot of DDM-solubilized membrane fractions showing 20 kDa CcmE. For (E) and (F), 70 ug total protein was analyzed. (G) Quantification of the results of heme staining (holoCcmE) and anti-CcmE immunoreactivity (total CcmE) from purified fractions from three independent experiments. (H) Quantification of the results of heme staining and anti-CcmE immunoreactivity from DDM-solubilized membrane fractions from three independent experiments. For (G) and (H), percent holoCcmE and total CcmE is relative to ΔGH, which has been set at 100%. Error bars denote SD. (I) Coomassie blue staining of purified GST-tagged CcmC from ΔGH delAB showing 48-kDa full-length GST-CcmC, 26-kDa GST, and 20 kDa CcmE. (J) Heme staining of purified GST-tagged CcmC showing co-purified 20-kDa holoCcmE. (K) Anti-GST immunoblot of purified GST-tagged CcmC showing 48-kDa full-length GST-CcmC and 26-kDa GST. (L) Anti-CcmE immunoblot of purified GST-tagged CcmC showing co-purified 20-kDa CcmE.