Binding of purified penton base to immobilized integrins. Ninety-six well plates were coated with purified integrins (0.1 μg/well; Chemicon International, Temecula, Calif.), and the plates were incubated with recombinant, WT, R340E mutant, and D288K mutant penton base proteins (at a concentration of 1 μg/ml in panels A and C and as indicated in panel B [10]) in the presence of different competitors. Followingincubation with anti-Ad anti-serum (10) and secondary incubation with an anti-rabbit horeradish peroxidase-conjugated antibody (Amersham Biosciences, Uppsala, Sweden), binding was analyzed by determining the optical density at 450 nm. Data are average values of results for three different experiments performed with duplicate samples + standard deviations. (A) Results are expressed as percentages of penton base binding values obtained in the absence of competitors. RGD, GRGDSP peptide (Invitrogen Italia SRL, Milan, Italy); RGE, GRGESP peptide at 392 μM (Gibco Invitrogen); LDV, PALLDVDA peptide at 392 μM (Primm, Milan, Italy); EDTA, 20 mM; α3, anti-α3 antibody P1B5 (Chemicon Int.); α5β1, anti-α5β1 antibody JBS5 (Chemicon International). White bars, integrin αvβ3; black bars, integrin α3β1. (B) Results are expressed as percentages of values obtained with 3 μg of the WT penton base protein/ml. Squares, WT penton base protein; circles, R340E penton base protein; triangles, D288K penton base protein. Solid lines, binding to αvβ3; dotted lines, binding to α3β1. (C) Results are expressed as percentages of penton base binding values obtained in the presence of CaCl₂ at a concentration of 1 mM. For Ca²⁺ results, CaCl₂ was used at 1 mM; for Mn²⁺ results, MnCl₂ was used at 2 mM; Mg²⁺ results, MgCl₂ was used at 2 mM. White bars, integrin αvβ3; black bars, integrin α3β1.

Transduction of mammalian cells in the presence of anti-integrin antibodies. A total of 10⁶ HeLa or SCC-25 cells were preincubated with anti-integrin antibodies (30 μg/ml) and then infected at a multiplicity of infection of 50 particles/cell with Adeno-GFP (kindly provided by N. La Monica, IRBM). Competition with anti-integrin antibodies of HeLa infection was performed in the presence of the anti-CAR antibody. GFP expression was determined by fluorescence-activated cell sorter analysis at 42 h postinfection. Data presented are mean values of results of duplicate samples + standard deviations. Antibodies: anti-αvβ3, LM609; anti-αvβ5, P1F6; anti-αv, I3C2; anti-α3, P1B5; anti-β1, JB1a; anti-α5β1, JBS5; anti-CAR, RmcB.

Mammalian cell adhesion to purified penton base, Ad, or fibronectin in the presence of anti-integrin antibodies. Ninety-six well plates were coated with purified penton base at 0.5 μg/well (10), Ad5 particles at 1.15 × 10¹⁰ particles/well (kindly provided by N. La Monica, IRBM, Pomezia [Rome], Italy), or fibronectin at 0.1 μg/well (Sigma Aldrich). A total of 10⁶ HeLa (ATCC CCL-2) or SCC-25 (ATCC CRL-1328) cells were added to the wells in the presence or absence of the different antibodies (20 μg/ml). Attached cells were quantitated by crystal violet staining and optical density determinations at 540 nm. Data are presented as average values of results of duplicate samples + standard deviations. Antibodies: anti-αvβ3, LM609; anti-αvβ5, P1F6; anti-αv, I3C2; anti-α3, P1B5; anti-β1, JB1; anti-α5β1, JBS5; anti-α6, MAb 1378; anti-CAR, RmcB. Except for RmcB and I3C2 (kindly provided by J. Bergelson and R. Klein,respectively), all the antibodies were purchased from Chemicon International. Competition with anti-integrin antibodies of HeLa binding to Ad was performed in the presence of the anti-CAR antibody. FN, coated fibronectin (0.1 μg/well; Sigma Aldrich); BSA, coated bovine serum albumin 3% (Sigma Aldrich).(A) Binding of HeLa to the penton base; (B) cell binding to Ad; (C) cell binding to fibronectin.