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. Author manuscript; available in PMC: 2014 Dec 1.
Published in final edited form as: Biochim Biophys Acta. 2013 Jul 18;1833(12):10.1016/j.bbamcr.2013.07.005. doi: 10.1016/j.bbamcr.2013.07.005

Figure 4. Lysine 135 within RhoA is the ubiquitin acceptor site for FBXL19.

Figure 4

A. MLE12 cells were co-transfected with FBXL19-V5 and RhoAN19-V5 or lysine mutant plasmids. Cell lysates were analyzed for RhoAN19-V5, mutants, FBXL19-V5, and β-actin by immunoblotting with antibodies to V5 tag and β-actin. B. MLE12 cells were transfected with RhoAN19K135R-V5 or RhoAN19K140R-V5 plasmid and then cells were treated with CHX for 0, 2, and 4 h. Cell lysates were analyzed for V5 tagged RhoAN19 mutants and β-actin by immunoblotting with V5 tag and β-actin antibodies. C. FBXL19-HA, RhoAN19-V5, and RhoAN19K135R–V5 were synthesized by a TnT system, and in vitro ubiquitinations were measured by incubation with E1, E2, ubiquitin, Cullin1, Skp1, and ATP, followed by V5 tag immunoblotting. Input lysates were analyzed for RhoAN19-V5, mutant, and FBXL19-HA by immunoblotting. Shown are representative blots from three independent experiments.