Fig. 4.

Ectodomain shedding of TNF-α is coregulated by GalNAc-T2 in Cell line models and in vivo. (A) Depiction of the TNF-α reporter construct containing GalNAc-T2 specific glycosylation and ADAM cleavage sites. (B) Western blot visualizing cleavage patterns in HepG2 WT, ΔT2, ΔT1, and ΔT2+T2 cells transiently transfected (48 h) with TNF-α reporter construct. (C) Visualization of AP activity in SDS-polyacrylamide gel in cell lysates and supernatants from HEK293 wild type and ΔT2 of full-length reporter and cleaved fragment. (D) Relative levels of shedding in HEK293 wild type, ΔT2, and ΔT3. Alkaline activity was measured in media and lysate. (E) Plasma levels (pg/mL) of TNF-α after LPS induction in WT and GalNAc-T2 KO mice. Statistical analyses were performed using the Student’s t test. Error bars show SDs.