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. 2013 Sep 12;288(44):31728–31737. doi: 10.1074/jbc.M113.495846

FIGURE 4.

FIGURE 4.

NEMO ubiquitination requires the presence of HOIP and HOIL-1L. A, linear chains are made by HOIP (top panel), whereas Strep-NEMO242–419 ubiquitination requires the addition of wild type HOIL-1L to the reaction (bottom panel). 1-h reactions were performed with 4 μm Strep-NEMO242–419 in 20 mm Hepes, pH 8, 50 mm NaCl, 1 mm MgCl2, 5 mm βMe. The figure shows two parts of a single Western blot. B, full-length HOIP and HOIL-1L ubiquitinate full-length GST-NEMO in a 1-h reaction. HOIP is slightly active in the absence of HOIL-1L. C, HOIP RING2 and LDD are essential for NEMO242–419 ubiquitination. The blots show the ubiquitin chain formation and Strep-NEMO242–419 modification with HOIL-1L and different HOIP mutants after 0, 0.5, 1, and 2 h.