Opticol human collagen type 1

HEK293 cells were grown on coverslips in 24-well plates coated with 50 μg/mL OptiCol™ human collagen type I (Cell Guidance Systems) to a confluency of 50–70%. Cells were transfected with pVLDLR_mGFP, pApoER2_mGFP, pmCherry-N1_ApoER2 + pVLDLR_mGFP or mGFP. After 24 h, 25 μl of concentrated RCM (cRCM) was added to the wells. Cells were incubated with cRCM at 37°C for 5 min, washed twice with cold PBS and fixed with 4% formaldehyde for 15 min at room temperature. Fixed cells were washed three times with cold PBS, incubated in blocking solution (1% BSA in PBS) for 30 min at room temperature and overnight with primary antibody (anti-Reelin, G10) at 4°C. On the next day, samples were washed three times with cold PBS and incubated with secondary antibody, goat anti-mouse IgG DyLight633 (ThermoScientific) for 1 h at RT. Afterwards, cells were washed three times with cold PBS and incubated 5 min in DAPI solution (5 μg/ml), washed again, incubated in quenching buffer (100 mM glycine) for 15 min. After the final wash with H₂0, coverslips were mounted using ibidi Mouting Medium (ibidi) and sealed with nail polish. Slides were analyzed using a confocal fluorescence microscope (laser-scanning microscope 700, Zeiss) and the corresponding ZEN software.

HEK293 cells were grown on 12-well plates coated with 50 μg/mL OptiCol™ human collagen type I (Cell Guidance Systems) to a confluency of 80%. Cells were transfected with pcDNA5myc_Dab1 and pClneo_ApoER2 or pcDNA5myc_Dab1 and pClneo_VLDLR or pcDNA5myc_Dab1, pClneo_ApoER2, and pClneo_VLDLR. After 24 h, cells were washed with PBS and kept in Imaging Medium (Hank’s Balanced Salt Solution, 2 mM Glutamine, 10 mM HEPES) for 30 min. Next, medium was changed to Reelin conditioned medium (RCM) or R3–6 was added to the cells (final concentration, 30 nM). After 20 min incubation at 37°C, cells were washed with cold PBS and lysed in NP-40 lysis buffer (150 mM sodium chloride, 1.0% Nonidet P-40, 10% glycerol, 20 mM Tris, pH 7.4) supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche), 1 mM EDTA, 0.05 M NaF and 1 mM Na₃VO₄. The lysates were centrifuged at 15,000× g for 15 min at 4°C. Protein concentration was measured with BCA Protein Assay Kit (Thermo Scientific) and 30 μg of proteins were separated on a 10% SDS-PAGE and immunoblotted for phospho-Dab1 (Ab pDab1, Cell Signaling) or GAPDH (Sigma Aldrich, St. Louis, MO, USA). Membranes were stripped and subsequently re-probed for the detection of Dab1 (AbD4).