Figure 4.

Immunoprecipitation experiments demonstrate that BDNF/TrkB signaling regulates PSD-95 palmitoylation through PLCγ and PKMζ in VC cultures. A, Palmitoylation of PSD-95 was increased in cultured VC neurons by BDNF (50 ng ml⁻¹) and decreased by TrkB block (1NM-PP1, 3 μm; N = 4). B, VC cultures with TrkB block (1NM-PP1, 3 μm) in the presence of BDNF (50 ng ml⁻¹) showed a reduction in PSD-95 palmitoylation ration compared to control (Bph-PP1, 3 μm; N = 4). C, VC cultures treated with blockers of the PLC pathway (U73122, 1 μm), but not the MAPK (PD98059, 50 μm) or PI3K (wortmannin, 100 nm) pathways, in the presence of BDNF (50 ng ml⁻¹) showed a decrease in PSD-95 palmitoylation (N = 4). D, VC cultures treated with the broad PKC blocker (chelerythrine, 2.5 μm) in the presence of BDNF (50 ng ml⁻¹) showed decreased palmitoylation (N = 4). E, VC cultures treated with PKMζ inhibitory peptide (a pseudosubstrate, 1 μm) or the scrambled peptide (1 μm) in the presence of BDNF (50 ng ml⁻¹) showed a decrease only with the pseudosubstrate (N = 4). Cultures in B–E were pretreated with BDNF (50 ng ml⁻¹). The signaling blocked is indicated for each lane below the blots. All immunoprecipitates (B–E) were resuspended in 33 μl of SDS-PAGE sample buffer; 30 μl were used for autoradiography [PSD-95 (³H Palm)], and the remaining 3 μl for immunoblotting [PSD-95 (IB)]. **P < 0.01; ***p < 0.001. Error bars represent SEM.