The intensity of GFP expression was quantitated using Image J. Chemotaxis assay Aggregation competent cells were prepared and stimulated with a glass capillary micropipette (Femtotip, Eppendorf, Hamburg, Germany) filled with 0.1 mM cAMP [56]. Time-lapse image series were captured and stored on a computer hard drive at 30 seconds intervals with a CCD camera. The DIAS software (Soltech, Oakdale, IA, USA) was used to trace individual cells along image series and determine cell motility parameters [57]. Subcellular fractionation Cells GSK126 purchase were collected by centrifugation and resuspended at a density of 2 × 108 cells/ml in MES buffer (20 mM 2-[N-morpholino]ethane sulfonic acid, 1 mM

EDTA, 250 mM sucrose, pH 6.5) supplemented with a protease inhibitor mixture (Roche Diagnostics, Mannheim, Germany). Cells were lysed

on ice by sonication and light microscopy was performed to ensure that at least 95% of the cells were broken. Cytosolic and particulate fractions were separated by ultracentrifugation (100,000 × g for 30 minutes). Alternatively the cell lysate was centrifuged to equilibrium on a discontinuous sucrose gradient atop an 84% (w/v) cushion. After centrifugation fractions were collected from the top and analyzed in Western blots or used for measurement of acid and alkaline phosphatase activities as described [52]. F-actin determination Chemoattractant Regorafenib induced F-actin formation in aggregation competent cells was quantitated as described [58]. Briefly, cells were resuspended at 2 × 107 cells/ml in Soerensen buffer and starved for 6 to 8 hours. Cells were stimulated with 1 μM cAMP and 50 μl samples were taken at various time points. The reaction was terminated by addition of 450 μl stop solution (3.7% formaldehyde, 0.1% Triton X-100, 0.25 μM TRITC-phalloidin in 20 mM potassium phosphate, 10 mM PIPES, 5 mM EGTA, 2 mM MgCl2 pH 6.8). After staining for 1 hour, samples were centrifuged for 5 minutes at 15,000 × g. Pellets were extracted with 1 ml methanol for 16 hours and fluorescence (540/565 nm) was read in a PTI fluorimeter

(Photon Technology Intl., Seefeld, Germany). Essentially the same procedure was used to determine the F-actin content of vegetative cells except that fluorescence values were Megestrol Acetate normalized to the total protein content of the samples as determined with the method of Lowry. Rac1 activation assay The Rac1 activation assay was performed as described [31]. Cells were starved for 6 to 8 hours in Soerensen buffer at a cell density of 1 × 107/ml, concentrated to 4 × 107/ml and stimulated with 1 μM cAMP. Aliquots were immediately removed and lysed in 5 × lysis buffer (50 mM HEPES pH 7.5, 2.5% Triton X-100, 500 mM NaCl, 100 mM MgCl2, 1 mM DTT) containing protease inhibitors at 4°C. The cell lysate was then mixed with glutathione-Sepharose beads previously loaded with bacterially expressed CRIB of Dictyostelium WASP fused to GST.