, Sunnyvale, CA) 100 nM in 10% DMEM), as previously described [3]. For all experiments except transfections, cells were harvested and analyzed on day 6. Indirect Immunofluorescence

and Phallacidin Staining Cells were cultured on FN coated 22 mm slides (BD Biosciences) which were situated in 6 well tissue culture plates, and inhibitors and blocking agents were added on day 3, as above. Slides were removed on day 6 and cells were fixed in 3.7% formaldehyde in LY2606368 PBS for 10 min. For phalloidin staining, slides were blocked in 1% bovine serum albumin (BSA) for 30 min and incubated in BODIPY-Phallacidin (green) or rhodamine phalloidin (red) (Molecular Probes) at room temperature for 20 min. For indirect Niraparib nmr immunofluorecence staining, cells were blocked in 5% BSA for one h, incubated with primary antibodies diluted in PBS 0.1% TRITON X-100, with overnight incubation at 4°C (Santa Cruz Biotechnology). Coverslips were washed and incubated with ALEXA FLOUR conjugated antibodies (Molecular Probes) and mounted on glass slides using Prolong Gold with Dapi (Molecular www.selleckchem.com/products/baricitinib-ly3009104.html Probes). Cells were viewed and photographed using a ZEISS Axiovert 200 M microscope fitted with an ApoTome Imaging system. Phalloidin staining was quantitated on 400 x magnification.

RhoA, GRAF and p190 Rho GAP indirect immunofluorescence slides were photographed at 630 x. Phospho-FAK Reverse transcriptase Y397 quantitation was performed on an Olympus BX20 fluorescence

microscope and an Olympus MagnaFire digital photographic system at 1,000x magnification. Immunoprecipitation, Rho GTP Assays and Western Blots For Immunoprecipitation, cells were incubated at clonogenic density on 10 cm fibronectin-coated plates, and cultured as above, harvested and lysed on day 6 in 1X modified RIPA buffer (Millipore) (approximately 2.5 million cells or 1 mg total protein). Pre-cleared lysates were subjected to immunoprecipitation using 10ug FAK antibody-conjugated agarose (Millipore) overnight at 4°C. Beads were washed in modified RIPA lysis buffer and collected by centrifugation at 10,000 X g, at 4°C. The beads were boiled in 1X Lamelli buffer (BIORAD) for 5 min and subjected to SDS PAGE for western blot analysis. For Rho GTP assays, equal numbers of cells were used because the dormant cells exhibited a larger cytoplasm to nucleus ratio and contained approximately 20% more protein than the growing cells (approximately 2.5 million cells, or 1 mg protein). Cells were harvested and lysed using 1X Mg Lysis buffer (Millipore). Rho GTP was immunoprecipitated using rhotekin RBD-agarose slurry (Millipore), which exclusively binds RhoGTP, according to the manufacturer’s instructions.