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Figure 4 | Molecular Neurodegeneration

Figure 4

From: LRP1 is critical for the surface distribution and internalization of the NR2B NMDA receptor subtype

Figure 4

LRP1ΔNPxY2 neurons show an altered phosphorylation of S1480 and Y1472 in NR2B receptor subunit. (A) Representative immunoblots demonstrate altered amounts of phosphorylated S1480 and Y1472 in NR2B receptor subunit at the surface of LRP1ΔNPxY2 neurons. Cortical LRP1ΔNPxY2 or WT neurons (DIV14) were subjected to the cell surface biotinylation. Biotinylated proteins were precipitated with NeutrAvidin agarose (surface). Membranes were probed with phospho-specific polyclonal NR2BpS1480 or NR2BpY1472 antibodies. Parallel, the same amounts of biotinylated proteins and input controls were loaded on the same SDS-Gel. The respective immunoblot was analyzed with a monoclonal NR2B antibody, to detect the entire amounts of biotinylated NR2B receptor at the cell surface for normalization of phospho-signals. (B) Protein expression was quantified by densitometric analysis of multiple blots. The intensities of cell surface signals were normalized to measured signal intensities for lysate controls (input). The obtained values for phosphorylated S1480 or Y1472 were normalized to signal intensities for the entire cell surface expression of NR2B receptor subunit. The values calculated for WT controls were set as 100%. The scale bars represent the mean percent change in the phosphorylation on S1480 or Y1472 of NR2B receptor subunit at surface of LRP1ΔNPxY2 neurons compared with WT controls ± S.E.M. for the phosphorylation of S1480 of LRP1ΔNPxY2 neurons an increase by 64% (p=0.04; n=4) and for the phosphorylation of Y1472 a decrease to 66% (p=0.03; n=4) of WT controls after a normalization to the entire NR2B cell surface expression. * p<0.05, Student´s paired t-test. (C) Representative Western blots demonstrate that the expression of casein kinase II or Fyn is not altered in LRP1ΔNPxY2 neurons. The phosphorylation levels of Fyn activation signal pY418 are not altered in LRP1ΔNPxY2 neurons. The membranes were probed with a phospho-specific polyclonal anti-Src pY418 antibody, which also reacts with Fyn pY418.

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