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An enzymatic dead mutant without the N-terminus also has tumor suppression activity (Nesiel-Nuttman et al., 2015).
Interestingly, the kinase dead mutant, D1994N, also displayed reduced GTPase activity, that may be indicative of its inability to autophosphorylate.
In addition, the mutant D224N was reported to be a kinase dead mutant.
As control a nuclease dead mutant FLAG-MUS81 D307A (D307A) was used.
Using a kinase dead mutant would seem to be more direct and convincing.
3) The solution phase assays especially with a dead mutant as substrate demonstrate the kinetic competence of intermolecular autophosphorylation.
Similar(33)
Likewise, IGF-1 activated ERK1/2 in cells expressing a kinase-dead mutant of the IGF-1 receptor.
However, LRRK2 greatly enhanced the level of the phosphorylated kinase-dead mutant of Rip2 (K47M) (Fig. 6A, lane 3 and 4).
The kinase-dead mutant of ULK1 (K46N) was made using site-directed mutagenesis kit (Stratagene).
To confirm the involvement of MSK1, we used fibroblasts transfected with a kinase-dead mutant of MSK1 (MSKD KD).
In contrast, expression of the deacetylase-dead mutant Sirt1-H363Y had minor effects.
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