Anti rac1 antibody clone 23a8

For immunofluorescence analysis, cells were cultured in 35 mm imaging dishes with an ibidi polymer coverslip bottom (ibidiTreat, Martinsfeld, Germany). Before staining, cells were washed with Dulbecco's Phosphate Buffered Saline (DPBS), fixed for 20 min with 4% paraformaldehyde at room temperature and permeabilized with 0.5% Triton X-100 for 15 min. Cells were blocked with 10% goat serum in DPBS containing 0.01% Triton X-100 and then stained with the indicated antibodies. Sister cultures in plastic flasks were lysed, and 13 μg of cleared cell extracts were examined by western blot analysis. As primary antibodies, Alexa Fluor 488-conjugated anti-MyHC monoclonal antibody (#53-6503-82, 5 µg ml⁻¹, MF20; eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) and anti-RAC1 antibody clone 23A8 (#05-389, 5 µg ml⁻¹; Sigma-Aldrich) were used. As secondary antibodies, Alexa Fluor 488-conjugated F(ab’)2-goat anti-mouse IgG antibodies (A-24920, 4 µg ml⁻¹; Thermo Fisher Scientific) were used for RAC1 detection. The actin cytoskeleton was labeled with phalloidin–TRITC (P1951, 50 µg ml⁻¹; Sigma-Aldrich). Cell nuclei were stained with DAPI (Sigma-Aldrich; 0.5 µg ml⁻¹).

The mouse monoclonal antibody 9B11 reactive against the c-Myc epitope (EQKLISEEDL) and the rabbit polyclonal antiserum reactive against human PLCγ₂ (sc-407) were obtained from Cell Signaling Technology and Santa Cruz Biotechnology, respectively. The rabbit polyclonal antiserum reactive against human Rac2 (sc-96) was purchased from Santa Cruz Biotechnology. The anti-β-actin antibody (clone AC-15) and the anti-Rac1 antibody (clone 23A8) were obtained from Sigma and Merck Millipore, respectively. The Rac inhibitor EHT 1864 and its inactive analog EHT 4063 were synthesized as described previously [41 ]. Human epidermal growth factor (EGF) (E9644) was from Sigma. ProGreen baculovirus vector DNA (A1) was purchased from AB Vector.