Fig. 2

Heparan sulfate modifications of BG promote ectodomain shedding. A Western blot of BG in CHO-K1 WT, 677, and 745 cells expressing FL-BG. B ELISA of BG from conditioned media collected from CHO-K1 WT, 677, and 745 cells expressing FL-BG. The concentration of shed-BG normalized to the total protein concentration of the cells is plotted by Mean ± SEM, (n = 6) *p < 0.05, **p < 0.005; One-way ANOVA followed by unpaired t-tests between CHO WT vs CHO 677, CHO WT vs CHO 745, and CHO 677 vs CHO 745 (p = 0.039 WT vs 677, p = 0.0018 WT vs. 745, p = 0.0456, 677 vs. 745). C Autoradiograph of samples after [¹²⁵ I]-TGF-β1 binding and crosslinking of cell surface receptors followed by immunoprecipitation using anti-BG antibody in indicated cells. D Western blot of BG from indicated HEYA8 cells after enzymatic treatment using chondroitinase-ABC (0.4U) and/or heparinase-III (50ng/mL) and quantification of western blot. The signal intensity of the BG-core band at 90 kDa was compared to the intensity of the larger GAG-modified BG band intensity spanning 100kDA to 250 kDa. Graph is plotted as %GAG to % core signal intensity normalized to the actin levels. E BG ELISA of the conditioned media collected from indicated HEYA8 cells. The concentration of shed-BG normalized to the total protein concentration of the cells is plotted by Mean ± SEM, (n = independent trials for Cntl = 8, FL-BG = 4, ∆GAG-BG = 4, S534A (CS-BG) = 4, S545A (HS-BG) = 5).; ****p < 0.0001, One-way ANOVA followed by unpaired t-test. p =  < 0.0001, FL-BG vs ∆GAG-BG, p < 0.0001, CS-BG vs. HS-BG, p = 0.0002, FL-BG vs. HS-BG)