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Blood, Vol. 112, Issue 8, 3242-3254, October 15, 2008
Functional analysis of the cytoplasmic domain of the integrin  1 subunit in endothelial cells
Blood Abair et al.
112: 3242
Supplemental materials for: Abair et al
Files in this Data Supplement:
- Figure S1. Loss and/or re-expression of the integrin α1 subunit does not alter fibronectin or fibrin-mediated signaling (JPG, 76.7 KB)
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(A) Endothelial cells null for integrin α1 or expressing the integrin subunit were plated on fibronectin for 0, 10 and 30 minutes. Cell lysates (20 µg/lane) were then analyzed by Western Blot for the levels of activated as well as total Akt, p38 MAPK and ERK. Images are representative of three independent experiments. (B) Phosphorylated and total kinase bands were quantified by densitometry analysis and the phosphorylated signal was expressed as phosphorylated kinase/total kinase ratio. Values are the mean ± SD of 3 experiments. Differences between α1KO and α1 full-length expressing cells (*), or cells at time 0 vs. cells on fibronectin (**) were significant (p<005). (C, D) The endothelial cells indicated were plated onto fibrin gels with or without 10 µM PD169316, 10 µM PD98059, and 5 µM LY294002. Tubulogenesis was then evaluated 12 hours after plating. Values are the means ± SD of one representative experiment. Differences between untreated vs. inhibitor treated cells (*) were significant with p<0.05.
- Figure S2. Integrin α1β1 is required for collagen-mediated p38 MAPK activation in endothelial cells (JPG, 38.4 KB)
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(A) 3.5 × 104 serum starved endothelial cells null for integrin α1 or expressing the integrin subunit were kept in suspension for 15 minutes with our without anti-human integrin α1 or anti-mouse integrin α2 antibodies (30 µg/ml final concentration) and then either left in suspension (Susp) or embedded into collagen I+IV gels (IIID) for 40 minutes. The gels were then run on SDS-PAGE and transferred. Membranes were cut at the level of the 50kDa molecular weight: the upper part of the membranes was incubated with anti-Akt antibodies, while the lower part was incubated with anti-phospho p38 MAPK antibodies. Images are representative of three independent experiments. (B) Phosphorylated and total kinase bands were quantified by densitometry analysis and the phosphorylated signal was expressed as phosphorylated kinase/total kinase ratio. Values are the mean ± SD of 3 experiments. (*) represents significant differences (p<005) relative to α1KO cells.
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