The peptide sequence aspartic acid-glycine-glutamic acid (DGE) is the collagen-binding domain to the http://www.selleckchem.com/products/Vandetanib.html ��2��1-integrin (33). The peptide sequence arginine-glycine-aspartic acid (RGD) is the laminin-binding domain to the ��2��1-integrin (28). The peptide sequence glycine-proline-arginine (GPR) is the fibrinogen-binding domain to the ��v��3-integrin (13). The peptide sequence GHRP served as a negative control. Ha31/8 is a function-blocking monoclonal antibody directed against the murine ��1-integrin subunit. The blocked cells then underwent attachment assay. Other blocked cells were inoculated with RRV for 1 h at 4��C, washed (to remove any unbound virus), warmed to 37��C, and incubated for 24 h. Viral yield was determined by FFA.

For each blocking assay, the sample size consisted of between three and five wells of cells per experimental condition, and each experiment was repeated at least three times. Flow Cytometry Direct immunofluorescent staining for the individual subunits of the integrins ��1��1, ��2��1, ��4��1, ��v��3, and ��x��2 was performed by using FITC-conjugated or R-phycoerythrin-conjugated monoclonal antibodies (BD Biosciences, San Jose, CA). Confluent monolayers were washed with PBS and were detached by using trypsin-0.75 mM EDTA for 10 min at 37��C. When testing for the ��4- and ��x-integrin subunits, cells were detached using PBS + 0.75 mM EDTA without trypsin because trypsin can degrade these integrins (15). H2.35 and mCl cells were resuspended to a concentration of 1 �� 106 cells/ml in FACS buffer (PBS + 0.

1% sodium azide) containing 10 ��g/ml of FITC-conjugated antibody and were incubated for 30 min at 4��C. Cells were washed with PBS + 0.1% sodium azide, pelleted by centrifugation, and resuspended in PBS + 1% formaldehyde. Background fluorescence was evaluated with isotype-control antibodies. Cells were analyzed (10,000 events per sample) by using a FACSCalibur dual-laser flow cytometer (BD Biosciences) and CellQuest software (BD Biosciences). To demonstrate that the ��2��1 heterodimer was present, indirect immunofluorescence staining by FACS analysis of the cells was performed by using 10 ��g/ml of rat anti-��2��1 primary IgG monoclonal antibody (BMA2.1; Chemicon International, Temecula, CA). A FITC-conjugated goat anti-rat IgG antibody (10 ��g/ml) was added, and the cells were analyzed as above.

Transfection of Small Interfering RNA mCl cells were seeded at a density of 5 �� 104 cells per well in 24-well plates and were incubated overnight at 37��C. Cells that were Anacetrapib 40% confluent were transfected with small interfering RNA (siRNA) or non-targeting siRNA (negative siRNA) according to the manufacturer’s protocol in 100 ��l of serum-free media with 1% l-glutamine and 3.35 ��l/well of X-tremeGENE (Roche, Basel, Switzerland).