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. Author manuscript; available in PMC: 2019 Aug 17.
Published in final edited form as: J Mol Biol. 2018 May 22;430(17):2802–2821. doi: 10.1016/j.jmb.2018.05.033

Figure 3.

Figure 3.

(a) The activity (picomoles of product per picomole of enzyme) of TR as ascertained through its ability to phosphorylate an optimal peptide substrate (pep-S) is stimulated by calmodulin (CaM) in the presence of Ca2+ (red). TR cannot phosphorylate pep-S in the absence of CaM (green). (b) TR (blue) and FL (red) show similar activity towards pep-S in the presence of Ca2+-CaM. The inset shows the kobs values, determined using the initial rate regime, for the two cases. Each datum represents an average of duplicate measurements. (c) Western blot analyses show lower levels of TR compared to FL in MCF-10A (eEF-2K−/−) cells following transient transfection, but comparable levels of eEF-2 phosphorylated on Thr-56 (all measurements in duplicate). eEF-2 levels in cells transfected with vector alone (CV), or in those expressing TR or FL, are shown. Pan-actin is used as loading control. The right panel shows the relative activities of TR and FL (as measured through Thr-56 phosphorylated eEF-2 levels; averaged over duplicate measurements) normalized to the cellular levels of each construct.