Skip to main content
. 2019 Aug 28;8(9):997. doi: 10.3390/cells8090997

Figure 7.

Figure 7

Blocking of N-glycosylation by tunicamycin leads to aberrant trafficking of Endoglin. (A–D) CHO or COS-7 cells were transfected with plasmids directing expression of rat FL-Endoglin (rFL-Eng), betaglycan (TβRIII), and rat soluble Endoglin (sol-Eng). A transfection with empty vector (pcDNA) served as control. Thereafter, cells were treated with tunicamycin (0.5 µg/mL, 24 h) or left untreated. (A) Cell extracts were prepared and analysed for expression of rFL-Eng and betaglycan. To monitor equal protein loading the membrane was reprobed with a β-actin specific antibody. (B) Cell lysates of COS-7 cells taken from (A) were incubated with ConA beads for precipation of glycosylated proteins. The ConA-bound fraction as well as the unbound fraction of the precipitates were then analysed by Western blot for rFL-Eng and betaglycan. (C) Supernatants of COS-7 cells transfected with vectors expressing rFL-Eng or sol-Eng were treated or not with tunicamycin (0.5 µg/mL, 24 h). Protein extracts were analysed by Western blot in the presence or absence of DTT (50 mM) for expression of sol-Eng. (D) Supernatants of COS-7 cells taken from (C) were incubated with ConA beads for precipitation of glycosylated proteins. The ConA-bound fraction as well as the unbound fraction of the precipitates were then analysed by Western blot for sol-Eng. (E) HSC Col-GFP cells were treated or not with tunicamycin (0.5 µg/mL, 24 h) and stimulated or not with TGF-β1 (1 ng/mL; 30 min). Thereafter, proteins were extracted and analysed by Western blot in the presence or absence of DTT (50 mM) for expression or activation of indicated proteins. Equal protein loading was demonstrated with a β-actin specific antibody. (F) Protein lysates of HSC Col-GFP (only TGF-β1 stimulated samples) taken from (E) were incubated with ConA beads for precipitation of glycosylated proteins. The ConA-bound fraction as well as the unbound fraction of the precipitates were then analysed by Western blot for the indicated receptor proteins. (G) HSC Col-GFP cells were transfected with a plasmid directing expression of rat soluble-Endoglin (sol-Eng) or empty vector control (pBK). Thereafter, cells were treated with tunicamycin (0.5 µg/mL, 24 h) or left untreated. Cell protein extracts were prepared and analysed in Western blot in the presence or absence of DTT (50 mM) for expression of sol-Eng.