Mab5366

Tissue pieces were prepared by adding protease and phosphatase inhibitor cocktail in RIPA buffer and centrifuging for 10 minutes at 12000x rpm to remove debris. Western blot was performed as previously described (22 (link)–24 (link)). From the lysates, protein concentrations were determined by the Lowry protein assay (500–0113, 500–0114, 500–0115; Bio-Rad). Equal amounts of protein were loaded into each lane of a 4–12% Tris gel (BioRad) and subjected to electrophoresis. After blotting, nitrocellulose-membranes (BioRad) were blocked for 1h (milk powder 5% in TBS/Tween 0.1–0.2%) and incubated with primary antibodies (ICAM-1, R&D Systems, AF796; E-Selectin, Santa Cruz, sc-137054; G10 anti-Reelin, made in-house; Reelin, Millipore, MAB5366; ApoER2, made in-house; GAPDH, Sigma-Aldrich, G8795; α-Tubulin, Millipore, CP08). Binding of secondary HRP-antibodies were visualized by ECL or ECL plus chemiluminescent (Amersham). After densitometric analyses with ImageJ, optical density values were expressed as arbitrary units and normalized for protein loading, as described in the figure legends.

Cell lysates or tissue pieces were prepared by adding protease and phosphatase inhibitor cocktail in RIPA buffer and centrifuging for 10 minutes at 12000 rpm to removed debris. From the lysates, protein concentrations were determined by the Lowry protein assay (500-0113, 500-0114, 500-0115; Bio-Rad). Equal amounts of protein were loaded into each lane of a 4-12% Tris gel (BioRad) and subjected to electrophoresis. After blotting, nitrocellulose-membranes (BioRad) were blocked for 1h (milkpowder 5% in TBS/tween 0.1-0.2%) and incubated overnight at 4°C with primary antibodies (pPI3K, Abcam, ab12135; PI3K, Upstate, 05-212; pAkt, Biosource, 44-622Z; Akt, Cell Signaling, 9272S; pIκBα, Abcam, ab12135; pP65, Cell Signaling, 3036S; P65, Cell Signaling, 4764S; pSTAT3, Cell Signaling, 9131; STAT3, Cell Signaling, 9139; E-Selectin, Santa Cruz, sc-137054; ICAM-1, R&D Systems, AF796; G10 anti-Reelin, made in house; Reelin, Millipore, MAB5366; GAPDH, Sigma-Aldrich, G8795), all diluted at 0,2 μg/ml. Binding of secondary HRP-antibodies were visualized by ECL or ECL plus chemiluminescent (Amersham). After densitometric analyses with ImageJ, optical density values were expressed as arbitrary units and normalized for protein loading to the total form of a protein (for the phosphorylated proteins) or to a housekeeping protein as indicated on the figures, as described in the figure legends.