mAb 8G9 to LMα5 chain strongly inhibits binding of isolated α3β1 integrin to immobilized laminins 511 and 521.

<p>A) α3β1 integrin binds rhLMs 511 and 521, but not rhLM411. B) Effect of mAbs to LMα5 chain on the binding of α3β1 integrin to rhLM511. C) Effect of mAbs to LMα5 chain on the binding of α3β1 integrin to rhLM521. Statistical analyses (Student's <i>t</i>-test) including mean and SD were calculated, as well as level of significance of integrin binding to rhLMs 411, 511 and 521 when compared to HSA (A), and effect of antibodies when compared to mouse IgG on integrin binding to rhLMs 511 (B) and 521 (C) (*, p<0.05; **, p<0.01; ***, p<0.001).</p

mAb 8G9 to LMα5 chain strongly inhibits α3β1/α6β1 integrin-mediated cell adhesion to laminin-511 and, together with LMα5 mAb 4C7, to laminin-521.

<p>A) KG1C glioma, B) BE melanoma and C) MDA-MB-231 breast carcinoma cells were used. Cells were allowed to adhere to surfaces coated with rhLMs 511 or 521 (20 μg/ml) for 1 hour at 37°C. Statistical analyses (Student's <i>t</i>-test) including mean and SD were calculated, as well as level of significance comparing antibodies to mouse IgG (*, p<0.05; **, p<0.01; ***, p<0.001).</p

mAb 8G9 to LMα5 chain strongly inhibits α3β1/α6β1 integrin-mediated cell migration on laminin-511 and, together with LMα5 mAb 4C7, on laminin-521.

<p>A) KG1C glioma, B) BE melanoma and C) MDA-MB-231 breast carcinoma cells were used. Cells were allowed to migrate for 18 hours at 37°C through 8-μm pore-membranes coated with rhLMs 511 or 521 (20 μg/ml) on the lower surface. Statistical analyses (Student's <i>t</i>-test) including mean and SD were calculated, as well as level of significance comparing antibodies to mouse IgG (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Immunohistochemical reactivity of mAb 8G9 in comparison to mAb 4C7 against LMα5 chain.

<p>(A) and (C), mAb 4C7; (B) and (D), mAb 8G9 against frozen sections of human kidney (A) and (B), and skin (C) and (D). The staining pattern of the two antibodies was indistinguishable. In kidney, the antibodies stained glomerular and tubular basement membranes, whereas in skin, reactivity with epidermal and vascular basement membranes was observed.</p

mAb 4E10 to a LMβ1 chain epitope near the globular domain of laminin-511 hinders the binding of mAb 8G9.

<p>A) mAb 4C7 and other LMα5 antibodies minimally affect the binding of mAb 8G9 to rhLM511. B) mAb 4E10 largely competes with the binding of mAb 8G9 to rhLM511. Statistical analyses (Student's <i>t</i>-test) including mean and SD were calculated, as well as level of significance of the effect of the mAbs on the binding of biotinylated-8G9 to rhLM511 when compared to mouse IgG. Competing antibodies were used at 50x higher concentration than biotinylated-8G9 (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Reactivity of novel mAbs to LMα5 chain as measured by ELISA, Western blotting and immunoprecipitation.

<p>A) Reactivity of the antibodies with rhLMs 411, 511 and 521 by ELISA. B) Reactivity of the antibodies with rhLM511 by Western blotting under reducing conditions. C) Ability of the antibodies to immunoprecipitate laminin-511 from A549 cells' conditioned medium and detection of the LMα5 chain by Western blotting with mAb 4B5. Bands of 300/350 kDa corresponding to LMα5 chain were detected with some of the antibodies.</p