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. 2004 Feb;24(3):1378–1386. doi: 10.1128/MCB.24.3.1378-1386.2004

FIG. 2.

FIG. 2.

Polyclonal antibodies directed against the ligand binding domains of ApoER2 and VLDLR induce Dab1 phosphorylation in primary embryonic neurons. (a) 293T cells were transfected with constructs encoding ApoER2 (ApoER2Δ4-6) (lanes 1 and 2) or VLDLR (lane 3) or with the empty vector (lane 4). The corresponding cell extracts were separated using SDS-8% PAGE under reducing conditions, and Western blotting was performed using Ab 20 (lane 1) or Ab 186 (lanes 2 to 4). Molecular size markers are shown at the left. (b) Primary mouse neurons were incubated with Reelin-conditioned medium, control medium, or antiserum 20 or 186 (1:1,000) (left panel). Neurons were stimulated with different concentrations of Ab 186 (right panel). Cells were processed for immunoprecipitation with anti-Dab1,and Western blotting was subsequently performed using antiphosphotyrosine (a-PY) or anti-Dab1 (a-Dab1) antibody as indicated. (c) Primary neurons were incubated with the preimmune serum from the rabbit producing Ab 186 or with Ab 186 alone or in the presence of an excess of soluble ligand binding domains of ApoER2 (AΔ4-6) or VLDLR (V1-8). Immunoprecipitation and Western blot analysis were performed as described for panel b. (d) Primary neurons from wt, ApoER2−/−, and VLDLR−/− mice were stimulated with Reelin, control medium, or Ab 186 (1:1,000). Immunoprecipitation and Western blot analysis were performed as described for panel b. (e) Primary mouse neurons derived from wt or VLDLR−/− mice were incubated with Reelin-conditioned medium, control medium, or serum from the rabbit producing Ab 187 at a dilution of 1:800. Cells were processed for immunoprecipitation with anti-Dab1 antibody, and Western blotting was subsequently performed using anti-PY and anti-Dab1 antibodies as indicated. Bands were scanned with a personal densitometer (Molecular Dynamics), and the antiphosphotyrosine signal was normalized to the anti-Dab1 band. The level of Dab1 phosphorylation induced by Reelin was set at 100% in each individual experiment. Relative intensities are shown boxed in the corresponding panels.