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. 2023 Aug 11;12:e86090. doi: 10.7554/eLife.86090

Figure 3. CaMKII holoenzymes do not mix during activation.

(A) Schematic representation of the experiment performed in panel (B) and possible outcomes. (B) Western blot detection of potential CaMKIIKD-AviTag incorporation in CaMKIIF394BzF holoenzymes. Lane 1 – CaMKIIF394BzF, lane 2 - CaMKIIF394BzF treated with UV, lane 3 - CaMKIIKD-AviTag, lane 4 - CaMKIIKD-AviTag treated with UV, lane 5 - CaMKIIF394BzF incubated with CaMKIIKD-AviTag, lane 6 - CaMKIIF394BzF incubated with CaMKIIKD-AviTag, then UV treated, lane 7 - CaMKIIF394BzF incubated with CaMKIIKD-AviTag and activation stimuli (Ca2+:CaM and Mg2+:ATP), lane 8 - CaMKIIF394BzF incubated with CaMKIIKD-AviTag and activation stimuli (Ca2+:CaM and Mg2+:ATP), then UV treated, lane 9 - CaMKIIKD-AviTag incubated with activation stimuli (Ca2+:CaM and Mg2+:ATP), lane 10 - CaMKIIKD-AviTag incubated with activation stimuli (Ca2+:CaM and Mg2+:ATP), then UV treated. Blue arrowheads indicate nonspecific, UV-induced, dimerization of CaMKIIKD-AviTag, independent of BzF.

Figure 3—source data 1. Uncropped blots used in panel Figure 3B.

Figure 3.

Figure 3—figure supplement 1. CaMKII holoenzymes do not mix during activation.

Figure 3—figure supplement 1.

(A) Coomassie stained gel of CaMKIIF394BzF and CaMKIIKD crosslinking used in Figure 3B. (B) Western blot detection of potential CaMKIIWT-AviTag incorporation in CaMKIIF394BzF holoenzymes. Blue arrowhead indicates a UV-induced, BzF-independent dimeric band of CaMKIIWT in the presence of 50 mM TCEP. (C) Crosslinking of CaMKIIF394BzF and CaMKIIWT-AviTag in the presence of 1 mM TCEP. Magenta arrowheads in lanes 3 and 4 indicate UV-induced, BzF-independent dimeric bands of CaMKIIWT, which are more abundant here than under conditions in panel B.
Figure 3—figure supplement 1—source data 1. Uncropped Coomassie stained gels and blots used in Figure 3—figure supplement 1.