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Blood, Vol. 108, Issue 8, 2608-2615, October 15, 2006
Soluble HLA-G1 inhibits angiogenesis through an apoptotic pathway and by direct binding to CD160 receptor expressed by endothelial cells
Blood Fons et al.
108: 2608
Supplemental materials for: Fons et al
Files in this Data Supplement:
- Figure S1. Purification of sHLA-G1 and sHLA-G1mono (PDF, 14.8 KB) -
The purity of the recombinant proteins was assessed by SDS-PAGE according to the method of Laemmli (1). Briefly, 3 µg of purified protein were loaded on a homogeneous 12% gel with 2-mercaptoethanol and were stained with Coomassie Brilliant Blue (A). Protein concentrations were established by the bicinchoninic acid assay (Pierce Chemical Co., Rockford,IL). For Western blot analysis of sG1 and sG1mono (B), the same amount of protein was subjected to SDS-PAGE and transferred to nitrocellulose. The membrane was blocked with 5% non-fat dry milk overnight at 4°C and incubated for 1 hour at room temperature with anti-HLA-G MEM-G1 mAb (Exbio, Praha, Czech Republic). The secondary antibody used was an anti-mouse IgG conjugated to horseradish peroxidase (Amersham Pharmacia Biotech). Proteins were visualized by ECL (Amersham Pharmacia Biotech)
(1) U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680–685.
- Figure S2. Western blot analysis of p85 cleaved PARP expression by trophoblast (PDF, 75 KB) -
SGHPL-4 trophoblast cells were incubated in the absence (control) or presence of sHLA-G1 (G1s, 0.1 µg/mL) or sHLA-G1mono (sG1mono, 0.1 µg/mL) for 50 h.
- Video 1 (MOV, 2.53 MB)
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SGHEC-7 endothelial cells were incubated with conditioned media from transfected cells with sHLA-G1 (1) or empty vector (2) and examined by time-lapse microscopy to assess the appearance of apoptotic morphology. Images were taken every 15 minutes from 0 to 45 hours. Endothelial cells incubated with sHLA-G1 conditioned medium become apoptotic. Endothelial cells incubated with control neo-conditioned medium expand rapidly and show low levels of apoptosis (t = 45 hours real time).
- Video 2 (MOV, 2.92 MB)
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