Figure 3
From: Neuralized family member NEURL1 is a ubiquitin ligase for the cGMP-specific phosphodiesterase 9A
NEURL1 but not NEURL1B promotes ubiquitination of PDE9A. (A) HA-tagged Ubiquitin (HA-Ub), PDE9A-V5His, Flag-NEURL1B, Flag-NEURL1, NEURL1-Flag, NEURL1-Rm-Flag and untagged NEURL1 were expressed in various combinations in HEK293-FT cells in the presence of proteasomal inhibitor MG-132. The proteins were immunoprecipitated using the indicated antibodies. Exogenously and endogenously ubiquitinated PDE9A were detected with HA and UbVU-1 antibodies, respectively. The higher molecular weight smear detected with HA and UbVU-1 antibodies in lanes 4, 5 and 9 indicates polyubiquitination of PDE9A. (B) PDE9A-V5His was expressed alone or together with either NEURL1-Flag or Flag-NEURL1 in HEK293-FT cells. Following immunoprecipitation with the indicated antibodies, PDE9A was detected in immunoprecipitates of endogenous ubiquitin with V5 antibodies and endogenous ubiquitin was detected in PDE9A immunoprecipitate with UbVU-1 antibodies. For clearer depiction of polyubiquinated PDE9A in the inputs of V5 immunoblots, the tonal range (i.e. levels) was changed differently for non-ubiquinated and polyubiquinated PDE9A species. The border for such differential level change is marked with a dotted line. IB, immunoblot; IP, immunoprecipitation.
