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. Author manuscript; available in PMC: 2012 Oct 21.
Published in final edited form as: Mol Cell. 2011 Sep 8;44(2):325–340. doi: 10.1016/j.molcel.2011.08.025

Figure 2. Determination of non-ubiquitin contribution to diGly sites.

Figure 2

A) Schematic of experiment.

B) Left, extracts from 8 hr Btz treated cells before and after mock or USP2cc treatment were immunoblotted with the indicated antibodies. Right, the total number of diGly sites (black bar) after Btz treatment and the number of sites in which their log2 ratio was decreased greater than 50% with USP2cc (grey bar).

C) Log2 ratios of all quantified diGly peptides from Btz treated cells either untreated (blue bars) or treated (red bars) with USP2cc.

D) Log2 ratios of all ubiquitin diGly sites (left) and representative known diGly modified proteins (right) from Btz treated heavy cells mixed with untreated light cells (blue bars) or light Btz treated, mock incubated and heavy Btz treated cells incubated with USP2cc post-lysis (red bars).

E) The fraction of the total quantified diGly sites with log2 ratios less than −1.0 (black bars) that were also unchanged after Btz treatment (grey bar) from cells treated as indicated.

F) Log2 ratios of representative peptides either known to be neddylated (CUL5) or putatively neddylated (NEDD8) from cells treated with Btz for 8 hours (blue bars), treated with Btz for 8 hours and subsequently treated with USP2cc post-lysis (red bars), untreated (green bars), or treated with MLN4924 for 2 hours (purple bars).

All error bars represent the SEM of multiple MS1 quantifications for the indicated site.