Rac1/2/3 Antibody #2465

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	-Rabbit (1:300)

Protocol tips
-Rabbit (1:300)

Publication protocol

Protein were extracted from cells by resuspension in RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Igepal, 5 mM EDTA (pH 8.0), 0.1% SDS supplemented with proteases inhibitors (Roche, Mannheim, Germany) followed by sonication and centrifugation (10,000 g/10 min) to clear out insoluble cell material. The bicinchoninic acid (BCA) assay (Interchim, MontiuconCedex, France) was used to quantify protein concentration. Proteins (50 μg of total cell lysates) were separated on either 4-12% Bis-Tris or 3-8% Tris-Acetate NuPAGE® gels (Invitrogen AB, Stockholm, Sweden). Proteins were subsequently blotted from the gels to nitrocellulose membrane (Hybond-C Extra, Amersham Biosciences) and blocked in Odyssey® blocking buffer TBST 1:1. Antibodies were added at + 4°C for 16h: Anti-Ephrin B3 (ab101699; 1:500), Anti-Ephrin A1 (ab 65072; 1:300) (both from Abcam, Cambridge Science Park, Cambridge, UK), Anti-EphA2 (#34-7400, 1:500; Invitrogen), Anti-phospho-Akt Ser473 (#9271, 1:1500), Anti-phospho-p44/42 MAP Kinase Thr202/Thr204 ERK (#9101, 1:500), Anti-phospho-p38 MAP Kinase Thr180/182 (#9211, 1:500), Anti-phospho Src Tyr416 (#2101, 1:500), Anti-vimentin (#5741, 1:1000) and Anti-Rac (#2465P, 1:300) (all from Cell Signaling MA, USA). Anti-E-cadherin antibody (#610181, 1:1000) was from BD Biosciences (MD, USA). Antibodies for EphA4 (#sc-921, 1:100), EphA3 (#sc-920, 1:500) and EphA5 (#sc-1014, 1:500) were all from Santa Cruz Biotechnology (TX; USA).
To control for loading differences, β-Tubulin (#T7816, 1:5000, Sigma Aldrich) was used. As secondary antibodies, anti-rabbit or anti-mouse antibodies (#5151 or #4408, 1:15000) from LI-COR Biosciences (Bad Homburg, Germany) were applied for 1 h. The resulting protein expression was examined and recorded by the use of the Odyssey® Sa Infrared Imaging System (LI-COR Biosciences).

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Manufacturer protocol

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