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. 2016 Jul 21;291(36):19132–19145. doi: 10.1074/jbc.M116.722637

FIGURE 6.

FIGURE 6.

USP36 and Nedd4-2 regulates each other ubiquitination. A, Nedd4-2 ubiquitination is reduced in presence of USP36. Lysates from HEK293 cells transfected with GFP-Nedd4-2, FLAG-USP36, or GFP-Nedd4-2 and FLAG-USP36 were subjected to immunoprecipitation (IP) using Nedd4-2 antibodies. Western blotting was performed to assess Nedd4-2 ubiquitination and Nedd4-2. The expression levels of USP36 and tubulin as a loading control are shown. A representative experiment is shown (n = 3). Note the reduced ubiquitination of Nedd4-2 when it is expressed together with USP36. B, USP36 expression does not affect Nedd4-2 levels. Lysates from HEK293 cells transiently transfected with Nedd4-2 and increasing concentrations of USP36 were subjected to Western blotting analysis to detect the amount of Nedd4-2. FLAG-USP36 protein levels from lysates are shown, and actin was used as a loading control. A representative experiment is shown (n = 3). C, USP36 polyubiquitination is modulated by Nedd4-2. Lysates from HEK293 cells transfected with GFP-Nedd4-2, USP36, or GFP-Nedd4-2 and USP36 were subjected to immunoprecipitation using FLAG antibodies. Western blotting was performed to assess USP36 ubiquitination and USP36. The expression levels of Nedd4-2 and actin as a loading control are shown. A representative experiment is shown (n = 2). Note the increased USP36 ubiquitination when Nedd4-2 is expressed together with USP36. D, Nedd4-2 expression down-regulates USP36 levels. Lysates from HEK293 cells transiently transfected with FLAG-USP36 and increasing concentrations of GFP-Nedd4-2 were subjected to Western blotting analysis to detect the amount of FLAG-USP36. GFP-Nedd4-2 protein levels from lysates are shown, and actin was used as a loading control. A representative experiment is shown (n = 3). Note the decrease in the levels of FLAG-USP36 with increasing amounts of GFP-Nedd4-2. E, Nedd4-2 ubiquitin ligase activity is required to down-regulate USP36 levels. Lysates from HEK293 cells transiently transfected with FLAG-USP36 and increasing concentrations GFP-Nedd4-2-C962S or YFP-Itch were subjected to Western blotting analysis to detect the amount of FLAG-USP36. GFP-Nedd4-2 protein levels and YFP-Itch are shown. Tubulin was used as a loading control. A representative experiment is shown (n = 3).