LTBP-2 blocks FGF-2-induced cell proliferation.

<p><b>A</b>. The effect of LTBP-2 on the bio-activity of FGF-2 was tested in a cell proliferation assay (see experimental). Human foreskin fibroblasts were treated with FGF-2 with and without follistatin (white columns), or FGF-2 and follistatin pre-incubated with 5 or 10 fold molar excess of full length LTBP-2 or fragment LTBP-2C F2 (cross-hatched). Negative controls (black columns), included cells only and cells incubated with follistatin, LTBP-2 or fragment LTBP-2C F2. Mean values ± S.D. from triplicate determinations. Note 5 fold molar excess of full-length LTBP-2 completely blocked FGF-2 induced cell proliferation (p = 0.0001) and 5-fold molar excess of fragment LTBP-2C F2 partially blocked the activity (p = 0.0001). <b>B</b>. Immunoblot analysis FGF receptor (FGFR1) phosphorylation. Human foreskin fibroblasts were treated for 2 hours with FGF-2 (10 ng / ml) only or with FGF-2 plus 10-fold molar excess of full length LTBP-2 (LTBP-2 FL) or fragment F2 (LTBP-2C F2). Control cells had no FGF-2 or LTBP-2 added. Cellular proteins were extracted and duplicate samples were analysed by SDS-PAGE and immunoblotting with anti-phospho-FGFR1 antibody, and anti-total FGFR1 antibody. Bands were visualised using the LI-COR Odyssey Infrared Imaging System. <b>C</b>. The band intensity was measured using ImageJ 1.48 software [National Institutes of Health (NIH), Bethesda, MD] and normalised to the internal β actin signal. The ratio of the phospho-FGFR1 to total FGFR1 value for each sample is expressed relative to the average FGF-2 only control value (= 100%). Note the strong FGFR1 activation by FGF-2 was substantially blocked by both LTBP-2 C and LTBP-2C F2 fragments. Mean values ± S.D. of duplicate lanes.