FIGURE 2:

Triacsin C does not generally inhibit the ubiquitin-proteasome system or protein secretion. (A) SDS lysates from HEK293 cells incubated with 1 µg/ml triacsin C for 16 h or 10 µM MG-132 for 6 h were analyzed by immunoblotting. (B) U2OS cells stably expressing Venus-DD were incubated with vehicle or 1 µg/ml triacsin C for 16 h, followed by emetine treatments for the indicated times. Venus fluorescence levels were monitored by flow cytometry and quantified as the percentage of the levels at time 0 h (n = 3). (C) HEK293 cells were incubated with vehicle or 1 µg/ml triacsin C for 16 h and then treated with 75 µM emetine for the indicated times. Where indicated, 10 µM MG-132 was added at the beginning of the emetine chase. The levels of the different forms of CD147 were assessed by immunoblotting of SDS lysates. (D) HEK293 cells expressing TTR-HA were treated with vehicle or 1 µg/ml triacsin C for 16 h. Cells were washed with PBS, and the medium was replaced with serum-free OPTI-MEM containing vehicle or 1 µg/ml triacsin C for the remaining 6 h. Lysates and TTR-HA immunoprecipitated from the media were analyzed by immunoblotting. (E) The levels of TTR-HA in the media were quantified from D and are presented as percentage of the levels in the control sample (n = 3). (F, G) The morphology of the ER, anti-KDEL (green) and the Golgi complex, anti-GM130 (green), in HeLa cells treated with vehicle or 1 µg/ml triacsin C for 16 h was analyzed by immunofluorescence microscopy. Nuclei were stained with DAPI (blue). Scale bar, 10 µm. Mat., mature; CG, core glycosylated; -CHO, deglycosylated. Error bars indicate SEM.