Fig. 5
From: Unraveling chain specific ubiquitination in cells using tandem ubiquitin binding entities
RIPK-Degrader 2 Mediates K48 Ubiquitination and Degradation of RIPK2. K562 cells (1.5 × 106 cells/ml) were treated with DMSO (vehicle control) or RIPK-degrader-2, PROTAC at indicated doses for 45 min. Cell lysates (30 µg) were separated on SDS-PAGE and probed with anti-RIPK2 antibody to detect RIPK2 degradation. Immunoblotting with anti-GAPDH antibody was used for loading control. Band intensities of RIPK2 normalized to GAPDH were used to quantitate RIPK2 degradation (%) and are shown below the western blot. Original western blot images are presented in Supplementary Fig. 5 (a) Lysates (15 µg/well) from RIPK degrader-2 treated cells were captured on Pan-selective TUBE. (b), K48-TUBE (c) and K63-TUBE (d) followed by detection with anti-RIPK2 antibody. Signal were developed using enhanced chemiluminescence (CL) substrate and luminescence was read using BMG LabTech Clariostar plate reader. Relative CL intensities were calculated by dividing raw CL signals from PROTAC treatment to the DMSO control and plotted using GraphPad Prism. Error bars represent standard deviation (n = 3).
