Figure 3

LRP1 and NR1/NR2B accumulate at cell surface of LRP1ΔNPxY2 neurons due to reduced endocytosis. (A) The Internalization rates of LRP1 and NMDAR were determined using a cleavable biotin assay in WT or LRP1ΔNPxY2 neurons. Two left panels (surface) demonstrate that MesNa buffer removes cleavable NHS-SS-biotin from the cell surface. Middle panels (internalized) show the amounts of internalized proteins after 15min at 37°C. Right panels (input) show the total amounts of proteins. Demonstrated blots represent 15min period of internalization. (B) The immunoblots were quantified by densitometric analysis. The internalization rates were calculated by comparing the values of internalized biotinylated proteins (internalized) to the values of respective biotinylated proteins at the cell surface of neurons in control dishes (Ctl.) and normalized to the signal intensities of the total amounts of the proteins (input). The internalization rates calculated for WT controls were set as 100%. The scale bars represent the mean percent change in internalization rates of proteins at the surface of LRP1ΔNPxY2 neurons compared with WT controls ± S.E.M. For the internalization rate calculated after 15min of internalization in LRP1ΔNPxY2 neurons for LRP1 a reduction by approx. 60% (p=0.01; n=5); for NR1 and NR2B a decrease by approx. 69% (p=0.001; n=5) and by approx. 70% (p=0.016; n=5) respectively. For the internalization rate of NR2A an insignificant decrease by 4% (n=4) in LRP1ΔNPxY2 neurons was calculated. (C) The internalization rates calculated in LRP1ΔNPxY2 neurons after 7 min of internalization. For LRP1 a reduction by 38% (p=0.043; n=4); for NR1 and NR2B a reduction by approx. 42% (p=0.04; n=4) and by approx. 39% (p= 0.044; n=5) was determined. For the internalization rate of NR2A an insignificant increase by 2% (n=4) in LRP1ΔNPxY2 neurons was calculated. The signal intensities of lysates were standardized to actin. * p<0.05; ** p<0.005 Student´s paired t-test.