stained with rhodamine- or FITC-conjugated phalloidin, and nuclei were stained with 49,6-diamidino-2-phenylindole. Following staining, cells were washed with PBS and then sealed under coverslips with a drop of Prolong anti-fade reagent. The fluorescent images were acquired on an inverted microscope using a 60x oil immersion objective. Digital images were obtained using MetaMorph software. For quantitative measurements of fluorescence at the leading PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19717433 edges of cells, a cell’s chemotactic leading edge was traced and the average fluorescence intensity was measured using ImageJ software. Relative fluorescence intensity was provided as the ratio of leading edge signal to whole cell signal in at least five different cells from each of three independent experiments. analyzed by IB as described previously, using secondary antirabbit IgG Alexa 680 Ab followed by visualization using an Odyssey Infrared Imaging System. Purification of GST/His-fusion proteins The growth of BL21-pLysS bacteria, induction of GST- or Histagged fusion proteins with isopropyl b-D-thiogalactopyranoside and protein isolation have 
been described previously. Rac and Cdc42 activation assays Identification of GTP-bound Rac or Cdc42 proteins from lysates using GST-PAK-PBD-beads was described previously. Protein-lipid binding assays Hydrophobic membrane strips containing eight phosphoinositides and seven related lipids were blocked with 3% BSA in PBS for 1 h at RT. The strip was then incubated for 1 h in blocking buffer supplemented with 1 mg/ml GST or GST-SSeCKS protein. After washing Seliciclib web thrice with PBS-T, the strips were incubated for 1 h at room temperature with mAb anti-GST Ab, washed thrice with PBS-T, incubated with horseradish peroxidase-conjugated anti-mouse Ig, washed thrice with PBS, incubated with Immunoblotting analysis RIPA cell lysates containing freshly added inhibitors and Complete Protease Inhibitor Mixture were Chemotaxis Suppression by SSeCKS/AKAP12 enhanced chemiluminescence reagent and autoradiographed. For the PIP-bead binding assay, 5 mg of GST or GST-SSeCKS proteins were incubated with 25 ml of PIP beads at RT for 1 h in blocking buffer, washed thrice with PBS-T and then the beads were analyzed by IB for GST. Statistical analyses All experiments were repeated at least thrice and then analyzed by un-paired t test and Tukey’s method for statistical significance using Prism v.3, with data shown as mean 6 S.E. Results Loss of SSeCKS leads to enhanced chemotaxis accompanying morphological changes at the leading edge Our group reported previously that SSeCKS suppressed chemotaxis in rat MATLyLu prostate cancer cells without affecting cell motility in monolayer wounding assays. Because chemotaxis assays typically measure parameters of individual cell migration whereas monolayer wounding reflects PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19717786 collective movements of cell sheets, this suggests that SSeCKS controls specific aspects of individual cell movement. To further address the role of SSeCKS in controlling chemotaxis, we compared motility of cells in Boyden chambers assays consisting of chemoattractants in the bottom chamber. SSeCKS-null -MEF displayed increased chemotaxis relative to matched WT MEF against serum or PDGF-BB . SSeCKS levels in DU145 and MDA-MB-231 are considered downregulated compared to untransformed epithelial cells, although to a greater extent in MDA-MB-231 cells, and thus, we sought to knockdown SSeCKS/AKAP12 levels in DU145 and overexpress SSeCKS in MDA-MB-231 cells. The siRNA-mediated k
