α9β1  (Bio-Rad)

Journal: The Journal of Cell Biology

Article Title: Polydom/SVEP1 binds to Tie1 and promotes migration of lymphatic endothelial cells

doi: 10.1083/jcb.202208047

Figure Lengend Snippet: Polydom binds to Tie1 via CCP20. (A) Recombinant integrin α9β1, Tie1, Tie2, EphrinB2, and Podoplanin were coated on microtiter plates at 5 μg/ml and then incubated with full-length Polydom (5 μg/ml). Polydom was allowed to bind to integrin α9β1 in the absence or presence of 1 mM MnCl 2 . The data represent means ± SD of triplicate determinations. (B) Titration curves of Tie1 and Tie2 binding to Polydom. Tie1 (open squares) or Tie2 (closed squares) at the indicated concentrations was allowed to bind to microtiter plates coated with full-length Polydom (10 nM). The data represent means ± SD of triplicate determinations. (C) Schematic representation of Polydom and its recombinant fragments. Polydom consists of multiple domains including von Willebrand factor type A (vWFA), Ephrin 2-like (Eph-like), hyaline (HYR), similar to thyroglobulin type 2 repeat (STT2R), EGF, and pentraxin (PTX) domains, and an array of CCP domains. (D) Polydom and its fragments were coated at 2 μg/ml on microtiter plates and incubated with Tie1 (2 μg/ml). The data represent means ± SD of triplicate determinations. (E) GST fusion proteins of CCP20 and CCP22 were coated at 2 μg/ml on microtiter plates and incubated with Tie1 (2 μg/ml). Pol-C and GST alone were included as positive and negative controls, respectively. The data represent means ± SD of triplicate determinations.

Article Snippet: A mAb against integrin α9β1 (Y9A2) was purchased from Millipore.

Techniques: Recombinant, Incubation, Titration, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Polydom/SVEP1 binds to Tie1 and promotes migration of lymphatic endothelial cells

doi: 10.1083/jcb.202208047

Figure Lengend Snippet: Integrin α9 is not involved in Polydom-induced LEC migration. (A) LECs were labeled with an anti-integrin α9β1 mAb (Y9A2; red line) or control mouse IgG (dashed line) and then subjected to flow cytometric analysis. The expression level of integrin α9β1 was significantly reduced in LECs transfected with ITGA9 siRNA. (B) Photographs of control and ITGA9 siRNA-treated LECs that migrated to the lower side of Transwell membranes. Bar, 200 μm. (C) LECs that migrated to the lower side of the membranes were counted under a microscope. The data represent means ± SD of three independent experiments each assayed in triplicate. NS, not significant ( n = 3). (D) Full-length Polydom and its Asp2638Ala/Glu2641Ala double-mutant (Polydom/AMMA) were assessed for their binding activities toward integrin α9β1. The data represent means ± SD of triplicate determinations. vWFA, von Willebrand factor type A; PTX, pentraxin. (E) Photographs of LECs that migrated to the lower side of Transwell membranes in the presence of Polydom or Polydom/AMMA (1 μg/ml) in the lower chamber medium. Bar, 200 μm. (F) Cells that migrated to the lower side of the membranes were counted under a microscope. The data represent means ± SD of three independent experiments each assayed in triplicate. NS, not significant ( n = 3).

Article Snippet: A mAb against integrin α9β1 (Y9A2) was purchased from Millipore.

Techniques: Migration, Labeling, Expressing, Transfection, Microscopy, Mutagenesis, Binding Assay