Figure 2.

Ipom-F inhibits protein secretion in a substrate-selective manner. (a) HepG2 cells were pre-treated either with DMSO, Ipom-F (100 nM) or tunicamycin (tun; 5 μg/ml) for 14 h or with BFA (2.5 μg/ml) for 2 h, followed by treatment in serum-free media for another 2 h in the presence of the compounds as shown in the schematic representation of the assay. Equal amounts of total protein from clarified cell lysates and serum-free media were analysed by immunoblotting for the indicated endogenous secretory proteins or the SNARE syntaxin-5 (Stx5). For the analysis of FGA, 2.5× more of the media sample than lysate sample was loaded to increase FGA signal sensitivity in the media. Tunicamycin treatment effectively prevented N-glycosylation (indicated by ψ) to yield non-glycosylated (0ψ) species. Endogenous α1AT can be observed as three protein bands. The upper bands represent the mature, post-ER forms of α1AT that contain complex N-glycans (reflected by heterogeneity in electrophoretic mobility), while the lower band corresponds to the immature, core-glycosylated form of α1AT located in the ER. The asterisk indicates a truncated form of AFP, previously described in HepG2⁷⁰ and perinatal rat liver⁷¹ cells, which becomes more prominent after treatment with stress-inducing compounds (see also Fig. 3a). Endogenous Stx5 exists as two isoforms, a 42 kDa-ER and a 35 kDa-Golgi isoform, that result from an alternative initiation of translation⁷². Note that the low levels of Stx5 recovered in media from BFA-treated cells may have been associated with BFA-induced loss of cell integrity. However, no signal from the abundant cytosolic tubulin was detected in these media, suggesting that the release of Stx5 in the media was most likely through unconventional secretion mechanism(s). Representative immunoblots are shown (full-length blots presented in Supplementary Fig. S6). (b) Quantification of protein levels in the media harvested from cells treated with Ipom-F or BFA (inset) as shown in (a). Secretion levels represent ratios of the protein signal in the media fraction to the corresponding signal in cell lysates (normalised against tubulin). Secretion levels were set to one in DMSO-treated cells. Values are mean ± s.e.m from two independent experiments.