Figure 5

NEURL1 mediates proteasomal degradation of PDE9A in HEK293-FT cells. (A) PDE9A-V5His was expressed either alone or together with various amounts (−, 0 μg; + , 1 μg; ++, 2 μg; +++, 3 μg) of Flag-NEURL1 or NEURL1-Flag or NEURL1-Rm-Flag in HEK293-FT cells. 24 hours after transfection, the cells were lysed and the lysates were immunoblotted with the indicated antibodies. (B) PDE9A-V5His was co-expressed with empty vector (‘negative control’) or NEURL1-Flag or NEURL1-Rm-Flag in HEK293-FT cells. Following transfection, the cells were first incubated for 16 hours in the presence of proteasome inhibitor MG-132 and then treated with 100 µM of protein synthesis inhibitor cycloheximide (CHX) for the indicated time periods. The lysates were immunoblotted with the antibodies indicated and the protein levels from two independent experiments were quantified. (C,D) PDE9A-V5His was expressed alone or together with NEURL1-Flag in HEK293-FT cells. 24 hours after transfection, the cells were treated with lysosomal inhibitor chloroquine or ammonium chloride (C) or with proteasome inhibitor MG-132 (D) at the indicated concentrations for 24 hours. The cells were lysed and subjected to immunoblotting with the indicated antibodies. In all experiments shown in A through D, immunoblotting with anti-GAPDH antibody was performed to demonstrate equal loading. IB, immunoblot.