Fig. 4.
Disruption of the Ub Y59-E51 loop inhibits cellular K48-polyubiquitination and triggers apoptosis. (A and B) Analysis of cellular K48-polyubiquitination in cells expressing of HA-Ub or HA-UbR54A/Y59A. (A) Immunoblot analysis. Odyssey Infrared Imaging System simultaneously images anti-HA reactive (green) and anti-K48 reactive signals (red). (B) Mass spectrometric analysis. See SI Appendix, Fig. S8C for details. (C–E) Ub replacement experiments. (C) Immunoblot analysis. CDT1 protein levels are indicated. The same protein size markers are used as in A. (D) Analysis of cell death by Annexin V staining. Graph represents an average of three independent experiments, with error bars for SD. (E) Cell cycle analysis. Quantification of cell cycle profile for each cell line is shown in SI Appendix, Fig. S9E.