Figure 4

HtrA2/Omi induces monoubiquitination rather than cleavage of its substrate UCH-L1 during TNF-induced necroptosis. A. Wild-type (WT) and HtrA2/Omi-deficient MEF were left untreated or stimulated for 16 h with 20 μM zVAD-fmk and 1 μg/ml CHX in the presence (to induce necroptosis) or absence (for control) of 100 ng/ml TNF before UCH-L1 was detected with a monoclonal antibody that recognizes the full-length 25-kDa form of UCH-L1 (mAB UCH-L1) or, on a parallel blot, with a polyclonal antibody to detect all cleavage fragments of UCH-L1 (pAb UCH-L1). An asterisk marks the 35-kDa band corresponding to the predicted size of monoubiquitinated UCH-L1. B. WT and HtrA2/Omi-deficient MEF were stimulated as in A, and additionally with 0.5 μM staurosporine for 8 h. Lysates were separated on 10–20% w/v Tris-Tricine gels (Biorad) to resolve low molecular weight proteins and immunoblotted with pAb UCH-L1. The blot was deliberately overexposed to visualize faint cleavage fragments. The 10-kDa UCH-L1 cleavage fragment generated by HtrA2/Omi during staurosporine-induced apoptosis is indicated (arrow, red box). C. WT and HtrA2/Omi-deficient MEF were treated with TNF/zVAD/CHX as in A for the indicated times and analyzed for proteins reactive with pAb UCH-L1 by Western blot. An asterisk marks the appearance of the 35-kDa band identical to the predicted size of monoubiquitinated UCH-L1. D. Lysates from WT and HtrA2/Omi-deficient MEF were incubated with 20 μM of an HA-tagged ubiquitin-derived probe (HAUbVME) in 50 mM Tris, 150 mM NaCl, pH 8.0 for 90 min at 37°C and subsequently analyzed by immunoblotting with HA antibody and reanalyzed with pAB UCH-L1. In panels A-D, detection of actin served as a loading control. E. An immunoprecipitation was performed using lysates from necroptotic WT MEF (treated with TNF/zVAD/CHX as in A) and an antibody for ubiquitin. Subsequently, UCH-L1 was detected by Western blot using pAb UCH-L1.