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. Author manuscript; available in PMC: 2014 Oct 1.
Published in final edited form as: Cell Signal. 2013 May 23;25(10):2047–2059. doi: 10.1016/j.cellsig.2013.05.012

Figure 3. Fbxl12 targets CaMKI for ubiquitination.

Figure 3

(A) In vivo ubiquitination assays. Ubiquitinated CaMKI was detected by immunoprecipitation of endogenous CaMKI followed by immunoblotting for ubiquitin after exposing cells to MG132. (B) MLE cells were transfected with V5-tagged CaMKI alone or co-transfected with HA-ubiquitin followed by MG132 exposure. Cells were then lysed and ubiquitinated V5-CaMKI was detected by immunoprecipitation with V5 antibodies followed by immunoblotting for HA antibodies. (C) In vitro ubiquitination assays. Purified SCF complex components were incubated with V5-CaMKI. The arrows show poliubiquitinated CaMKI. (D) Diagram of CaMKI deletion mutants. Arrows indicate putative ubiquitin acceptor sites. (E) Immunoblot analysis of cells transfected with V5-CaMKI FL or truncated V5-tagged CaMKI mutant plasmids followed by exposure to MG132. (F) Immunoblot analysis of cells transfected with V5-CaMKI FL or V5-tagged CaMKI point mutant plasmids (K59R and K110R) followed by exposure to MG132. (G) Cells transfected with V5-tagged (V5-CaMKI FL) or V5-tagged CaMKI point mutant plasmids (V5-K59R and V5-K110R) were exposed to MG132 and then processed for immunoprecipitation using V5 antibodies followed by immunoblotting with anti-ubiquitin antibodies. (H) Determination of CaMKI half-life using cycloheximide in cells expressing either CaMKI FL or the CaMKI K59R mutant plasmids with quantification (I). (J) Levels of CaMKI in the cells co-transfected with CaMKI FL or CaMKI K59R mutant plasmids after ectopic expression of Fbxl12 plasmid (data are from n=3 experiments). *P < .01 versus CHX-treated CaMKI FL.