Adding glucose, and Ub reexpression is often induced by adding copper to the growth medium (Fig. 1A). To validate Ub silencing and reexpression, Ub, UbUbWT, and UbUbK0 yeast strains had been treated with glucose and copper. As shown in Fig. 1B, Ub expression was effectively suppressed, and each UbWT and UbK0 were markedly expressed. To assess monoubiquitination in human cells, we utilised a modification of a previously described Ub replacement model in human cultured cells (21). Briefly, endogenous Ub is silenced in U2OS cells by a Ub-specific tetracycline-induced shRNA (shUb), and either HA-UbWT or HA-UbK0 is expressed following infection with an adenoviral vector (Fig. 1A). To evaluate Ub silencing efficiency, we monitored Ub and Ub conjugates level in U2OSshUb cells following tetracycline therapy. As demonstrated in Fig. 1Ci, the degree of both Ub and Ub rotein conjugates were considerably decreased. Within the endogenous Ub-silenced cells, both HA-UbWT and HA-UbK0 had been efficiently expressed and assembled into highmolecular-mass conjugates following adenoviral expression (Fig. 1Cii). It needs to be noted that the pattern of conjugation seems related for both UbWT and UbK0 expression. This is possibly as a result of a lot of substrates with a broad range of molecular mass that happen to be conjugated, and from the possibility that a lot of of them are modified by a number of monoubiquitinations. To demonstrate Ub replacement utilizing an added process, we quantified Ub using mass spectrometry (MS). As shown in Fig. S1A, tryptic digestion of UbWT and UbK0 yields each typical and differential MS-detectable peptides. To assess Ub replacement in yeast, we treated UbUbK0 cells with glucose and copper, and quantified Ub-derived peptides by MS.(3-(4-Hydroxyphenyl)acryloyl)glycine Data Sheet As illustrated in Fig.165617-59-4 In stock S1B,E4640 | www.PMID:25105126 pnas.org/cgi/doi/10.1073/pnas.Fig. 1. Replacement of endogenous Ub by UbK0 in yeast and mammalian cells. (A) Workflow describing Ub silencing and reexpression (see a detailed description under Experimental Procedures). (B) Ub replacement in yeast cells. Ub, UbUbWT, and UbUbK0 yeast cells have been treated for Ub silencing and Ub reexpression as indicated. Yeast cells have been analyzed by trichloroacetic acid (TCA) lysis followed by SDS/PAGE and Western blotting (WB) utilizing the indicated antibodies. (C) Ub replacement in human cells. (i) Ub silencing. To silence Ub, U2OSshUb cells had been treated with tetracycline (1 g/mL) for the indicated occasions. (ii) Ub reexpression. Following Ub silencing, cells had been infected with viral vectors expressing UbWT or UbK0. In each panels, lysates have been analyzed by means of SDS/ Web page followed by WB using the indicated antibodies.UbK0 was markedly more abundant than endogenous Ub. To evaluate UbK0 expression in human cells, we overexpressed HAUbK0 by means of adenoviral infection utilizing increasing multiplicities of infection (MOIs). As displayed in Fig. S1C, UbK0 expression level was MOI dependent and substantially exceeded the level of endogenous Ub. Taken together, these data demonstrate the effectiveness of our Ub replacement techniques and recommend that our experimental systems are appropriate for studying protein monoubiquitination.Systematic Identification of Monoubiquitination-Dependent Proteasome Substrates. To recognize substrates which might be degraded following mono-ubiquitination, we replaced Ub with either UbK0 or UbWT (as a control). We then used anti -e-GG immunoprecipitation (Fig. S2) to enrich and quantify by MS GlyGly-modified peptides derived from tryptic digestion of ubiquitinated prote.