Anti integrin α2 clone p1e6

IGF1 receptor (IGF1R) expression was evaluated fluorometrically by the PE-conjugated anti-Human CD221 (IGF1R; clone1H7; BD Pharmingen) monoclonal antibody. Mouse IgG1 (clone P3.6.2.8.1; Thermo Fisher, Dreieich, Germany) was used as the control isotype. In ongoing experiments, the tumor cells were activated with LONG^®R3IGF-1 (100 ng/mL) and adhesion, chemotaxis, and the integrin α2 (clone 12F1-H6), αV (clone 13C2), and β1 (clone MAR4) surface expression was analyzed. Adhesion to immobilized collagen and chemotaxis was also evaluated when tumor cells were serum starved for 12 h and stimulated with IGF-1 and simultaneously blocked with anti-integrin α2 (clone P1E6) or anti-integrin β1 (clone 6S6), both mouse mAb (all obtained from Merck Millipore, Burlington, MA, USA). BTT3033 (Bio-Techne, MN, USA), a selective inhibitor of the integrins α2β1, or Cyclo(RGDyK) (Selleckchem, TX, USA), targeted against integrin αVβ3, were also employed to determine whether cross-communication between IGF-evoked Akt signaling and integrin expression exists. HepG2 cells were activated with IGF and integrin α2 and β1 expression were measured by flow cytometry and compared to the integrin expression in HepG2 cells treated with the AKT inhibitor MK-2206 (Selleckchem, TX, USA).

UMUC3 and RT112 cells were incubated for 60 min with 10 μg/mL function-blocking anti–integrin α2 (clone P1E6), anti-integrin α3 (clone P1B5), anti–integrin α6 (clone NKI-GoH3), anti-integrin β1 (clone 6SG), or anti-integrin β4 (clone ASC-8; all: MerckMillipore). Controls were incubated with cell culture medium alone. Subsequently, tumor cell adhesion to immobilized collagen as well as chemotaxis was analyzed as described above.