Figure 4
From: Neuralized family member NEURL1 is a ubiquitin ligase for the cGMP-specific phosphodiesterase 9A
NEURL1 promotes polyubiquitination of PDE9A via lysines K27, K29 and K33 of ubiquitin. (A) Wild-type HA-tagged ubiquitin, K0 ubiquitin (i.e. ubiquitin mutant whose lysine residues had been substituted for arginine) and the various K only ubiquitin mutants were co-expressed with either PDE9A-V5His only or with PDE9A-V5His and NEURL1-Flag in HEK293-FT cells in the presence of proteasome inhibitor MG-132. The proteins were immunoprecipitated and immunoblotted using the indicated antibodies. (B) Wild-type HA-tagged ubiquitin, K0 ubiquitin and the various K only ubiquitin mutants were co-expressed with either PDE9A-V5His only or together with NEURL1-Flag in HEK293-FT cells in the absence of proteasome inhibitor MG-132. The lysates were immunoblotted using the indicated antibodies. (C) Quantification of NEURL1-mediated degradation of PDE9A in the presence of different ubiquitin mutants when overexpressed either alone or with NEURL1. Coomassie-normalised levels of PDE9A in cells co-expressing NEURL1 and the respective ubiquitin mutant were divided with the respective levels of PDE9A in cells where NEURL1 was not overexpressed. The calculated ratio in cells overexpressing the K0 mutant was arbitrarily set as 1. The average of 5 independent experiments is shown, with error bars representing SEM. Statistical significance was calculated relative to the ratio in cells expressing K0 ubiquitin. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed paired t-test. IB, immunoblot; IP, immunoprecipitation.
