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When exposed to tert-butyl hydroperoxide, survival of the lon-1 mutant was impaired. In addition, production of several virulence factors such as BosR, RpoS, and OspC was elevated in the mutant. These phenotypes were restored when lon-1 mutation was complemented. Finally, we created a lon-1(S714A) mutant and found that this mutant failed to infect mice, suggesting that the proteolytic activity of Lon-1 is essential for bacterial infection. Taken together, these results demonstrate that Lon-1 is required by B. burgdorferi to infect animal