Skip to main content
. 2021 Feb 22;22(4):2172. doi: 10.3390/ijms22042172

Figure 5.

Figure 5

NHK mannose trimming in the presence of EDEM3 mutants. (A) NHK processing by the EDEM3 constructs. EDEM3-KO cells were co-transfected with NHK and EDEM3 mutants and processed for Western blotting. The resulting membranes were incubated with rabbit anti-α-1 antitrypsin (A1AT), rat anti-HA and mouse anti-actin antibodies. (B) Kifunensine treatment hinders EDEM3 constructs mannosidase properties. EDEM3-KO cells were co-transfected with EDEM3 constructs and NHK. Kifunensine (25 µM) was added to the cell media and equal aliquots of cell lysates were separated by SDS-PAGE and processed for WB with rat anti-HA antibody. Calnexin (CNX) was used as loading control. (C) Pulse-chase analysis of NHK intra and extracellular fate. EDEM3-KO cells were co-transfected with plasmids encoding for NHK and EDEM3 constructs. After immunolabeling, lysates (upper panel) and cell media (lower panel) were immunoprecipitated with rabbit anti-A1AT antibodies ON. The isolated immunocomplexes were separated by SDS-PAGE and visualized by autoradiography. (D) Graphic representation of NHK secretion rate as percent of total amount of three independent experiments (mean ± SEM).