Figure 5.

Deleting the N-terminal region of PSD-95 increases Src-mediated NMDAR tyrosine phosphorylation. (A) Upper: immunoprecipitations with anti-NR2 from lysates of HEK293 cells (300 μg) cotransfected with NR1/2B, Y527F Src or K295R Src, and wild-type (WT) PSD-95 or Δ(14–54) PSD-95. Immunoprecipitated NR2B was sequentially probed with anti-pY and anti-NR2B. Blots shown are representative of three separate experiments. Lower: densitometric quantification from three experiments, one of which is represented above. Band intensity was quantitated as mean gray value and the ratio of pY NR2B to total NR2B was calculated. Bars correspond to mean (±s.e.m.) ratios normalized to ratios obtained for cotransfection of NR1/2B with Y527F Src and wild-type PSD-95. (^*P<0.0005, t-test versus Y527F Src and wild-type PSD-95). Inset: representative 5-point standard curve (R²=0.99, P<0.001) derived from serial dilutions of forebrain proteins. (B) Upper: immunoblots with lysates of HEK293 cells transfected with NR1/2B, Y527F Src, and WT PSD-95 or Δ(14–54) PSD-95 (left). In each lane, 30 μg of protein were loaded. Blots were sequentially probed for NR2B, Src, PSD-95 and GAPDH, which served as a loading control. Results are representative of three separate experiments. Immunoprecipitations were carried out from the cell lysates (300 μg) with anti-NR2 (right). Proteins were sequentially probed with anti-NR2B, anti-Src and anti-PSD-95. Results are representative of three separate experiments. Lower: densitometric quantification from three experiments, one of which is represented above. Bars correspond to mean (±s.e.m.) band intensity, which was quantitated as mean gray value (P>0.4, t-test versus WT PSD-95). (C) Upper: immunoprecipitations with a pan anti-Src antibody from lysates of HEK293 cells (300 μg) transfected with Y527F Src plus no PSD-95, WT PSD-95 or Δ(14–54) PSD-95. Immunoprecipitated Src was immunoblotted with anti-pY416 Src (upper) and anti-Src (lower). Input was 30 μg of proteins from cells cotransfected with Y527F Src and Δ(14–54) PSD-95. Positions of IgG heavy chains are indicated. Results are representative of three separate experiments. Lower: densitometric analysis from three experiments, one of which is represented above. Bars correspond to mean (±s.e.m.) band intensity, which was quantitated as mean gray value (^*P<0.01, t-test versus no PSD-95 or versus Δ(14–54) PSD-95).