The protein concentration of the lysate was determined by BCA protein assay

or chemotaxis, SSeCKS’ ability to PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19717844 suppress chemotaxis did not require its Src scaffolding activity. Taken together, our data strongly suggest that SSeCKS suppresses chemotaxis by differentially organizing PIP3, Rhofamily GTPases and their regulatory proteins, Src and regulators of actin cytoskeletal remodeling at the leading edge. Materials and Methods Cell culture C57BL/6 wild type and SSeCKS2/2 primary mouse MEF were derived from E13.5 embryos as described previously. Production of the MEF was performed under protocol 963 M approved by the Institutional Animal Care and Use Committee of Roswell Park Cancer Institute. Pregnant females were euthanized by sodium pentobarbital overdose, and to minimize suffering, embryos were placed in ice-cold PBS followed by decapitation. Cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol with initial passages plated on gelatinized T25 flasks. Human cancer cell lines, DU145 or MDA-MB231, were cultured in RPMI 1640 or DMEM, respectively, containing 10% FBS. Antibodies and Reagents The primary antibodies used include rabbit polyclonal Abs: SSeCKS, GAPDH, PKC-f, Akt; mouse monoclonal Abs: His6, glutathione S-transferase, Rac1 Cdc42, PIP3, PIP2, Frabin; goat polyclonal Ab: Par6. The following secondary Abs were purchased from Invitrogen: anti-rabbit Alexa Fluor 568 and Alexa Fluor 488, anti-mouse Alexa Fluor 568 and Alexa Fluor 488. Rhodamine-labeled phalloidin and fluorescein isothiocyanate -conjugated phalloidin were from Invitrogen. Human AKAP12-si-RNA and mouse Fgd4 siRNA were from Santa Cruz and Thermo Scientific, respectively. Other reagents include LY294002, SKI-606, digitonin, Human epidermal growth factor , LipoD293, Lipofectamine 2000 and Oligofectamine. Plasmids and transfection pcDNA3.1-SSeCKS was produced by PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19717794 transferring the 5.8 Kb EcoRI order Aglafoline fragment from pBluescriptSSeCKS. Chemotaxis Suppression by SSeCKS/AKAP12 3 Chemotaxis Suppression by SSeCKS/AKAP12 chemotactic WT cells predominantly display lamellipodia whereas those of KO cells predominantly display filopodia-like extensions. Left panels- phase contrast microscopy of chemotactic cells. Open-head black arrows, filopodia; closed-head black arrows, lamellipodia; white arrows, chemotaxis direction. Middle and right panels- IFA staining of SSeCKS or F-actin in WT or KO MEF. Arrows, chemotaxis direction. Scale bar, 10 mm. F, Fraction of chemotactic WT or KO cells with leading edge lamellipodia or filopodia. Error bars, S.E. of 5 visual fields with at least 10 cells/field in three independent experiments. , p,0.02, , p,0.005. G, Percentage of chemotactic WT or KO cells in Panel F with,1, 13 or.3 filopodia/leading edge. , p,0.005. H, Induction of lamellipodia formation in MDA-MB-231 cells transfected with SSeCKS-GFP, as shown by IFA for GFP or F-actin, or following quantification. Arrows, chemotaxis direction.Cells were transiently transfected using LipoD293 and 5 mg plasmid DNA according to the manufacturer’s instructions, and lysates were produced using radioimmunoprecipitation assay buffer 1840 h later. siRNA transfections were carried out in 6-well dishes using 20 50 nM siRNA in Lipofectamine 2000 or Oligofectamine according to manufacturer’s instructions. Lysates were prepared 4872 h later. Chemotaxis assays Boyden chamber. Chemotaxis assays were performed in 24well modified Boyden chambers as described