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. Author manuscript; available in PMC: 2015 Jul 18.
Published in final edited form as: Biochem Biophys Res Commun. 2014 Jun 10;450(1):550–554. doi: 10.1016/j.bbrc.2014.06.019

Figure 1. CaMKII does not mediate phosphorylation or activation of CYLD in isolated PSDs under Ca2+-free conditions.

Figure 1

PSD fractions were pre-incubated for 15 min with or without ATP and CN21, an inhibitor of both Ca2+-dependent and autonomous forms CaMKII, as indicated. Samples were subsequently incubated for indicated times with K63-linked tetra ubiquitin (Ub4) to test DUB activity. Western immunoblots show CYLD phosphorylation at S-418 using a phospho-specific antibody (p-CYLD) (top panels) in comparison to total CYLD levels (middle panels). DUB activity was monitored as the rate of degradation of Ub4, as shown in the coomassie gel stain (bottom panels). Addition of ATP promoted CYLD phosphorylation and increased Ub4 breakdown and inclusion of the CaMKII inhibitor CN21 had no effect on either of these reactions. Two independent experiments yielded similar results.