Flag epitope m2 clone

Membrane-permeable C3 transferase was from Cytoskeleton, Inc. Cytochalasin D, nocodazole, lysophosphatidic acid and FITC-phalloidin were from Sigma. All other pharmacological reagents were from Calbiochem. The following mouse monoclonal antibodies were used: to V5 epitope (GKPIPNPLLGLDST; [34 (link)]; Invitrogen); to FLAG epitope (M2 clone; Sigma); to DsRed (Clontech or Abcam); to β-tubulin (Sigma); to TSP1 (A6.1; Neomarkers). Rabbit monoclonal to COMP (EPR6289(2)) was from Abcam, FITC-conjugated goat polyclonal IgG to V5 tag was from Abcam. Alkaline phosphatase-conjugated secondary antibodies were from Applied Biosystems and FITC-conjugated secondary antibodies were from Sigma. COS7, SW480 human colon carcinoma and rat chondrosarcoma (RCS) cells (A.T.C.C.) were all cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) in a humidified, 5% CO₂ incubator at 37°C.

Whole-cell lysates were separated by SDS-PAGE, transferred to Hybond-ECL membranes (Amersham) and blocked with 5% nonfat dry milk. Membranes were probed with primary antibodies the FLAG epitope (M2 clone; Sigma) or β-actin (AC-15; Sigma Aldrich), then with HRP-conjugated secondary antibodies (Jackson ImmunoLaboratories) and detected with enhanced chemiluminescence (ECL; Pierce). Band intensities were quantified with Image Studio Lite (LI-COR) software.