Anti rac1 23a8

Anti-RhoA (3L74, dilution 1:500), anti-Cdc42 (Cat. No. 17–299, dilution 1:500), anti-Rac1 (23A8, dilution 1:2,500) and anti-GAPDH (6C5, dilution 1:20,000) antibodies were from Millipore (Schwalbach, Germany), anti-GFP (A10262, dilution 1:500), anti-chicken Alexa Fluor 488 (A11039, dilution 1:1,000), anti-rabbit Alexa Fluor 555 (A21430, dilution 1:1,000) antibodies were from Life Technologies (Darmstadt, Germany), and Horseradish peroxidase (HRP)-conjugated anti-MBP (Cat. No. E8038, dilution 1:5,000) was from New England Biolabs. Anti-GST (Cat. No. 27-4577-01, dilution 1:2,000) was from GE Healthcare, HRP-linked anti-mouse antibody from Rockland Immunochemicals (Limerick, PA) and HRP-conjugated anti-rabbit antibody from New England Biolabs.

Standard western blot assays were used to analyze protein expression in cells. The following antibodies were used for assays: anti-Flag-M2 (F1804, Sigma; 1:20,000 dilution), anti-β-Actin (A5441, Sigma; 1:10,000 dilution), anti-Myc (9E10, Roche, Indianapolis, IN; 1:1000 dilution), anti-HA (3F10, Roche; 1:1000 dilution), anti-Rac1 (23A8, Millipore, Billerica, MA; 1:5000 dilution), anti-p-PAK (Ser199/204) (09–258, Millipore; 1:1000 dilution), anti-PAK (07–1451, Millipore; 1: 1000 dilution), anti-p53 (FL393, Santa Cruz, Dallas, TX; 1:2000 dilution), anti-Tiam1 (sc-872, Santa Cruz; 1:2000 dilution), anti-VAV1 (sc-8039, Santa Cruz; 1:1000 dilution). The anti-GLS2 antibody (1: 1000 dilution) was prepared as previously described (Hu et al., 2010 (link)). To increase the sensitivity of the GLS2 antibody, endogenous GLS2 in cells was pulled down by IP and detected by western blot assays. The band intensity was quantified by digitalization of the X-ray film and analyzed with the ImageJ software.

Embryos were lysed in a buffer consisting of 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 2 mM EGTA, 25 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 1 % Nonidet P-40, 10 mM NaF, 1 mM vanadate, 1 mM DTT, and 1× Protein Inhibitor Cocktail (Sigma). Extracts were then centrifuged, and supernatants were collected. Anti-Myc (A14, Santa Cruz), anti-GFP (JL8, Clontech), anti-Rac1 (23A8, Millipore) or anti-α-tubulin (DM1A, Sigma) antibodies were used as primary antibodies, and anti-mouse IgG HRP-conjugated (1:10,000; GE healthcare) and anti-rabbit IgG HRP-conjugated (1:10,000; GE healthcare) were used as secondary antibodies.