FIGURE 3.

Reelin down-regulates VLDLR levels and induces receptor ubiquitination in hippocampal neurons. Hippocampal neurons were stimulated with 1 μg/ml Reelin under various conditions as indicated below. Immunoblotting was performed using specific antibodies against VLDLR and β-actin (used as a control). Left panels, immunoblots; right panels, quantification. A, cells were stimulated for 24 h. Values are means ± S.D. (n = 3). *, p < 0.05 for Reelin versus the control (C). B, the addition of 0.3 μm bafilomycin A₁ (Baf), which inhibits lysosomes, blocked the degradation of VLDLR induced by Reelin. Values are means ± S.D. (n = 3). ***, p < 0.001 for bafilomycin A₁ + Reelin versus Reelin; **, p < 0.01 for bafilomycin A₁ versus the control; *, p < 0.05 for Reelin versus the control and bafilomycin A₁ + Reelin versus the control. C, Reelin down-regulates VLDLRs that were increased by 50 ng/ml BDNF. Values are means ± S.D. (n = 3; ANOVA p value, 0.0023; F, 39.36; Tukey's test). **, p < 0.01 for BDNF versus the control. D, cells were stimulated in the absence or presence of 1 μm GW3965 (GW). Note the decrease in VLDLR levels by GW3965 and Reelin. Values are means ± S.D. (n = 3; ANOVA p value, 0.0001; F, 50.93; Tukey's test). ***, p < 0.001 for Reelin versus the control and for GW3965 + Reelin versus the control; **, p < 0.05 for GW3965 versus the control and for GW3965 + Reelin versus GW3965. E, VLDLR ubiquitination. Lysates were prepared from control hippocampal neurons and from cells stimulated for 24 h with Reelin. Immunoprecipitations (IP) were done employing 2 μg of anti-VLDLR monoclonal antibody, followed by immunoblotting as described under “Experimental Procedures.” Left panel, ubiquitin species bound to VLDLR in the immunoprecipitate; right panel, total amount of VLDLRs in lysates. Note the increase in the degree of ubiquitination of VLDLR in Reelin-treated cells. A typical experiment is shown and was repeated three times.