Figure 5

Knock-in mutation into NPxY2 motif of LRP1 results in stronger binding of NR2B with LRP1. (A) The representative immunoblot demonstrates the lacking of interaction of NR1 with LRP1 in a co-immunoprecipitation experiment. Equal amounts of total lysates (80µg) were used for immunoprecipitation with a polyclonal LRP1-specific antibody (1704) [50]. The membranes were probed with a monoclonal NR1-specific antibody. (B) The representative immunoblot demonstrates a stronger interaction of NR2B with LRP1 in cortical LRP1ΔNPxY2 neurons. After an immunoprecipitation with a LRP1-specific antibody (1704) [50] the membranes were probed with a monoclonal NR2B-specific antibody. (C) A treatment with CIP results in a complete dephosphorylation of tyrosines Y4507 in wild type LRP1 and Y1472 in NR2B, while serine 1480 in NR2B shows a residual phosphorylation. The equal amounts of CIP-treated lysates and untreated controls were analyzed. Membranes were probed with a polyclonal phospho-specific LRP1pY4507 antibody and polyclonal phospho-specific antibodies NR2BpY1472 or NR2BpS1480. (D) The dephosphorylation of proteins in cell lysates of WT neurons results in a reduction of NR2B co-immunoprecipitated with LRP1, while a dephosphorylation of LRP1ΔNPxY2 lysates has no effect on the binding of NR2B to LRP1. CIP-treated neuronal lysates derived of WT or LRP1ΔNPxY2 cortical cultures or untreated controls were used for co-immunoprecipitation. The co-purified proteins were detected with a monoclonal NR2B-specific antibody. (E) The binding of PSD95 with LRP1 is not directly affected by LRP1ΔNPxY2 knock-in mutation. Representative immunoblot demonstrates the equal amounts of PSD95 protein co-precipitated with LRP1 in cell lysates of LRP1ΔNPxY2 neurons or WT controls. (F) The dephosphorylation of proteins in cell lysates of WT neurons results in an increase of PSD95 co-immunoprecipitated with LRP1, while a dephosphorylation of proteins in LRP1ΔNPxY2 lysates has no effect on the binding of PSD95 to LRP1. Immunoblots shown are representative results from at least three independent experiments.