Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Loss-of-function thrombospondin-1 mutations in familial pulmonary hypertension

doi: 10.1152/ajplung.00282.2011

Figure Lengend Snippet: A: THBS1 gene, including chromosomal neighbors. All exons (rectangles) and introns (lines) are depicted. Exons 7–9 (type I repeats) were resequenced for this project. Direction of gene translation is per arrows. B: trimeric THBS1 protein (TSP1). N, globular region N (NH2-terminal); C, procollagen homology domain; TSR1, 2, and 3, thrombospondin type I repeats; E1, 2, 3, EGF homology (type II) repeats; CaG, calmodulin homology (type III) repeats; G, globular domain (COOH-terminal). The location of the missense mutation found in TSR1 is designated with an asterisk (*). Exon sizes are relative to each other but are exaggerated for display.

Article Snippet: After washing, the membrane was incubated with a mouse anti-human TSP1 antibody (Abnova; H00007057) at 1 μg/ml in Tween-TBS-3% nfdm for 2 h at 22°C.

Techniques: Mutagenesis

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Loss-of-function thrombospondin-1 mutations in familial pulmonary hypertension

doi: 10.1152/ajplung.00282.2011

Figure Lengend Snippet: The 362Asn TSP1 mutation displays loss-of-function characteristics on latent TGF-β1 activation in the mink lung epithelial cell bioassay. A: latent TGF-β1 activation by synthetic 13-amino acid TSR1 peptides centered on the 362Asn TSP1 locus. At all concentrations wild-type TSR1 (362Asp) is a more potent activator compared with mutant TSR1 [362Asn, which activates more than scrambled peptide (SCR) only when ≥5 nM]. 362Asp effects were always greater than SCR effects (*P < 0.05). B: trimeric recombinant TSP1 (rTSP1) is a more potent activator of TGF-β1 (pM effects). The mutant isoform is weaker at all concentrations compared with the wild-type isoform (*P < 0.01, **P < 0.001). Activation is expressed as fold change compared with vehicle (zero). Symbols indicate statistical significance by ANOVA or unpaired t-tests. Results represent summed means ± SD of ≥4 experiments.

Article Snippet: After washing, the membrane was incubated with a mouse anti-human TSP1 antibody (Abnova; H00007057) at 1 μg/ml in Tween-TBS-3% nfdm for 2 h at 22°C.

Techniques: Mutagenesis, Activation Assay, Bioassay, Recombinant

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Loss-of-function thrombospondin-1 mutations in familial pulmonary hypertension

doi: 10.1152/ajplung.00282.2011

Figure Lengend Snippet: 362Asn mutation displays loss-of-function properties on TSP1 inhibition of human pulmonary artery smooth muscle cell (HPASMC) proliferation at 72 h. Full-length rTSP1 was used for all studies. Only wild-type 362Asp rTSP1 had significant inhibitory effects. Stimulatory effects on proliferation were seen with both isoforms at low concentrations. Results are expressed as means ± SD, summed results of 4 experiments. *P < 0.05 **P < 0.01 vs. vehicle (zero) values.

Article Snippet: After washing, the membrane was incubated with a mouse anti-human TSP1 antibody (Abnova; H00007057) at 1 μg/ml in Tween-TBS-3% nfdm for 2 h at 22°C.

Techniques: Mutagenesis, Inhibition