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. Author manuscript; available in PMC: 2014 Sep 5.
Published in final edited form as: Cell. 2014 May 22;157(5):1061–1072. doi: 10.1016/j.cell.2014.03.046

Figure 4. Zinc dependence of M7CK binding to zinc-finger domain (ZfD)-containing proteins.

Figure 4

A. HA-tagged M7CK was co-expressed with FLAG-tagged interacting proteins in 293T cells. The cell lysate was split into equal aliquots and supplemented with 20 μM TPEN and 50 μM of the indicated ions. M7CK was IP’d with αHA and the co-IP’d proteins probed with αFLAG.

B. FLAG-tagged ZfD-containing proteins were co-expressed in 293T cells with HA-tagged M7CK. [Zn2+] was adjusted by variation of [EGTA] and [ZnSO4]. M7CK-HA was IP’d with HA-agarose; ZfD protein binding was detected in the αFLAG WB.

C [Zn2+] dependence of M7CK binding to GST-RYBP ZfD. Purified GST fusion with RYBP fragment (a. a. 22–47 comprising the RYBP-ZfD) was combined with purified M7CK-L in the presence of varying [Zn2+] and pulled down by glutathione agarose (Coomassie-stained gel).

D. Quantification of M7CK binding shown in C. Values of integrated pixels of the bound M7CK image were normalized to maximum binding at 10 μM [Zn2+]. Fitted equation and parameters shown.