FIGURE 3:

Triacsin C impairs ERAD substrate glycan trimming. (A) HEK293 cells were pretreated with vehicle or 1 µg/ml triacsin C for 16 h, followed by 75 µM emetine for the indicated times. Where indicated, 5 µg/ml kifunensine and 5 µM CB-5083 were added at the beginning of the emetine chase. SDS lysates were separated on large-format SDS–PAGE gels and analyzed by immunoblotting to visualize the different CD147 glycoforms. A darker exposure of the CD147(CG) bands is provided to facilitate visualization of the different trimmed glycoforms. (B, C) The relative levels of untrimmed CD147(CG) (B) and trimmed CD147(CG) (C) were quantified from A and are presented as percentage of the levels at time 0 h (n = 3). (D) Lysates from cells treated as in A were incubated with PNGase F as indicated and analyzed by immunoblotting. Mat., mature; CG, core glycosylated; -CHO, deglycosylated. Error bars indicate SEM.