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. Author manuscript; available in PMC: 2010 Sep 8.
Published in final edited form as: Biochemistry. 2009 Sep 8;48(35):8483–8490. doi: 10.1021/bi901058g

FIGURE 5.

FIGURE 5

Guanabenz causes a mechanism-based ubiquitination of nNOS. A, guanabenz acts in the heme/substrate binding cleft. nNOS was preincubated with Hsp70/40 under conditions required for enzyme activity (see Methods) and in the presence of 100 μM guanabenz, 100 μM D-NNA, or 100 μM L-NNA as noted at the top of the figure. The samples were then incubated with the ubiquitinating enzyme mixture and Western blotted with anti-nNOS. B, guanabenz-dependent ubiquitination requires nNOS enzymatic activity. nNOS was preincubated with Hsp70/40 under conditions for full enzyme activity or minus calmodulin (no enzyme activity) in the presence or absence of guanabenz as noted at the top of the figure. The samples were then incubated with the ubiquitinating enzyme mixture and Western blotted.