Fig. 2.

Quantitation of unanchored poly-ubiquitin chain formation by ICP0 using near-IR imaging. In vitro reaction mixtures containing E1, E2 (UBE2D1), and E3 (GST-ICP0.241) were activated through the addition of wild-type ubiquitin (±Ub) and incubated at 37 °C for the specified time (mins). Reaction mixtures were resolved by SDS–PAGE and analyzed by multiplex western blotting in conjunction with near-IR imaging for the formation of unanchored poly-ubiquitin chains (mAb P4D1 and Dylight anti-mouse 800; green) and ICP0 auto-ubiquitination (pAb 3678 and Dylight anti-rabbit 680; red). (A) Representative image of a scanned immunoblot showing RGB (left-hand panel) and corresponding single channel grayscale images (anti-ICP0 and anti-ubiquitin; middle and right-hand panels, respectively). Unmodified ICP0, auto-ubiquitinated ICP0 (Ub-ICP0), and unanchored poly-ubiquitin chains (Poly Ub) are highlighted. (B) Regions of interest (ROI; dashed boxes right-hand panel in A) relating to unanchored poly-ubiquitin chain formation were quantified and normalized with respect to the 90-min time point in the presence of ubiquitin within individual experiments. Scatter plot depicts the mean intensity for each time point. Bars represent the standard errors of the means (SEMs) from three independent experiments. Regression analysis, R², and ½ V_max values were calculated using Sigmaplot (Systat Software, Inc).