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. 2013 Jun 6;153(6):1312–1326. doi: 10.1016/j.cell.2013.05.014

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© 2013 ELL & Excerpta Medica.

This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.

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Figure S5 — Stable Inducible OTULIN Cell Lines and Controls, Related to Figure 4

(A) Western-blotting analysis of T-REx293 cells stably expressing a doxycycline-inducible MRGS6His-tagged OTULIN construct.

(B) NF-κB luciferase activity of OTULIN-overexpressing cells in response to LUBAC coexpression. Error bars represent standard deviation of experiments performed in duplicate. p values are given to indicate significance and asterisks indicate mean values set to 1.

(C) Quantification for the experiment shown in Figure 4C.Black bars represent the number of HeLa cells in which p65 translocated into the nucleus while white bars represent the number of cells in which p65 was excluded from the nucleus after 30 min of TNFα stimulation (20 ng/ml) in cells transfected with the indicated OTULIN variants. n = 30 for each condition. Error bars represent standard deviation from the mean from duplicate measurements.

(D) Control to Figure 4C. HeLa cells were transiently transfected with indicated plasmids and analyzed in immunofluorescence with indicated antibodies without any prior TNFα stimulation (see Extended Experimental Procedures). Scale bar = 10 μm.

(E) Input levels of transfected proteins for pulldown of GST-tagged NEMO variants of Figure 4D in control or OTULIN overexpressing T-REx293 cell lines (see Extended Experimental Procedures) after western blotting with the indicated antibodies.

(F) Pulldown of GST-tagged wild-type NEMO after coexpression with LUBAC, in control or OTULIN overexpressing T-REx293 cell lines. Western blotting with indicated antibodies reveals polyUb on NEMO (arrowhead), which is lost when OTULIN is coexpressed. This was the first experiment showing that NEMO is modified by a Met1-linked polyubiquitin chain of a distinct length and is shown to support the results of Figure 4D.

(G) Immunoprecipitation of endogenous OTULIN (see Extended Experimental Procedures) coprecipitates endogenous SHARPIN before and after TNFα stimulation (100 ng/ml) as revealed by western blotting with the indicated antibodies.^*, nonspecific band; ^**, heavy chain.