Hras c 20

Antibodies against β-actin and K-Ras were obtained from Sigma, H-Ras (c-20) and N-Ras (F155) from Santa Cruz Biotechnology, Ras and Ral-A from Upstate, M-Ras from Thermo Scientifics, R-Ras, AKT, p-AKT (S473), ERK1/2, p-ERK1/2 (T202/T204) from Cell Signaling Technology, and RASA1 from Epitomics. Active Ras-GTP and Ral-GTP levels were determined using Ras and Ral Activation Assay Kits (Millipore) respectively, following the manufacturer's instructions.

To obtain hippocampal tissue, animals were anesthetized with isoflurane after which they were decapitated and tissue was quickly isolated. Tissue was homogenized in 0.1 M Tris-HCl (pH 6,8), 4% SDS supplemented with protease and phosphatase inhibitor cocktails (Sigma). The concentration of protein was adjusted to 1 μg/μl. 15 μg was used for Western blot analysis using 18-wells Criterion XT Bis-Tris precast gels (Bio-Rad). When using 26-wells gels, 10 μg protein was loaded. Primary antibodies used were HRAS (HRAS (C20), Santa Cruz Biotechnology), ERK (p44/42 MAPK, Cell Signaling), phospho-ERK (Ph-p44/42 MAPK, Cell Signaling). Actin was always included as a loading control (Actin, Millipore). Blots were probed with IR Dye secondary antibodies (IR Dye 680LT or 800CW, LI-COR) and visualized using LI-COR Odyssey infrared imager. For quantitative analysis, Odyssey V3.0 software was used.

Cell lysates were separated on 15% polyacrylamide gels and transferred onto nitrocellulose for 2 h at 250 mA, followed by blocking with 5% (w/v) nonfat dried milk for 1 h. Blots were incubated with the appropriate primary antibody with dilution according to the manufacturers’ instructions [beta-actin(Mab AC-40, Sigma; dilution 1:5000); PAK2 (Cell signaling 2608, dilution 1:1000); phospho-S144/141-PAK1/2 (Abcam ab40795; dilution 1:2000); Rac1 (BD Transduction Laboratories 610650, clone 102; dilution 1:1000); Rac1(Millipore 05-389, clone 23A8; dilution 1:1000); Ras(Mab27H5, Cell Signaling 3339, dilution 1:200); H-Ras (C20, SantaCruz sc-520, dilution 1:200); in buffer B (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5 mM KCl, 0.05% (w/v) Tween 20] for 18 h and subsequently for 2 h with a horseradish peroxidase-conjugated secondary antibody (mouse: Rockland 610-1034-121; dilution 1:3000; rabbit Rockland 611-1302; dilution 1:3000). For the chemiluminescence reaction, ECL Femto (Pierce) was used. The signals were analyzed densitometrically using the KODAK 1D software.