Fig. 1: mGlu7 is neddylated in heterologous cells and primary cortical neurons.
From: Neddylation is required for presynaptic clustering of mGlu7 and maturation of presynaptic terminals
a HEK 293 T cells were cotransfected with myc-mGlu7 and HA-NEDD8 or HA-NEDD8ΔGG and treated with 1 μM MLN4924 or vehicle (DMSO) for 6 h. Thirty-six hours after transfection, cell lysates were immunoprecipitated with an anti-myc antibody. Western blotting was performed with anti-HA or anti-myc antibodies. *, nonspecific bands; MLN, MLN4924. b HEK 293 T cells were cotransfected with myc-mGlu7, HA-NEDD8, and UBC12 C111S-His or UBC12 WT-His. Immunoprecipitation and western blotting were performed as shown in panel a. An open triangle indicates the UBC12 C111S-NEDD8 complex. *, nonspecific bands. c Neddylation of endogenous mGlu7 is blocked by MLN4924. Primary cortical neurons (DIV14) were treated with 1 μM MLN4924 or vehicle for 6 h. Cell lysates were immunoprecipitated with anti-mGlu7 antibody and subjected to western blotting using the indicated antibodies. d, e Agonist stimulation reduces mGlu7 neddylation. Primary cortical neurons (DIV14) were treated with 400 μM L-AP4 for 5 min at 37 °C. Cell lysates were immunoprecipitated with an anti-mGlu7 antibody (d) or an anti-NEDD8 antibody (e). The immunoprecipitates were analyzed by western blotting. The bar graph with scatter plots shows the mean ± SEM (mGlu7 HMW, vehicle: 1.00 ± 0.06, L-AP4: 0.59 ± 0.01; mGlu7 LMW, vehicle: 1.00 ± 0.01, L-AP4: 0.32 ± 0.06; n = 3, *p < 0.05, Student’s t test).
