Fig. 3
From: Unraveling chain specific ubiquitination in cells using tandem ubiquitin binding entities
High throughput ELISA assay to differentiate linkage-specific ubiquitination of RIPK2. THP1 cells were pre-treated with DMSO (vehicle control) or indicated doses of Ponatinib for 30 min followed by treatment with water (control) or 200 ng/ml L18-MDP for 30 min. Cell lysates were incubated on pan-selective TUBE (a and b), K63-selective TUBE (c and d) or K48-TUBE (e and f) coated plates. Ubiquitinated RIPK2 was detected using an anti-RIPK2 antibody and an HRP-conjugated secondary antibody followed by enhanced chemiluminescence (CL) signal generation. Relative increase in ubiquitination (top panel, relative CL intensity) and percent inhibition and IC50s of RIPK2 ubiquitination by Ponatinib (lower panel) were plotted using GraphPad Prism (n = 3). Error bars represent standard deviation. One-way ANOVA, ****p < 0.0001, ***p = 0.0004, ns = non-significant.
