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. 2021 Jun 18;10:e60660. doi: 10.7554/eLife.60660

Figure 6. M1-linked/linear ubiquitin chains generated in cells and conjugated to NEMO in vitro are sensitive to hydroxylamine treatment.

Figure 6.

(A) Hydroxylamine treatment of M1 linkage-containing poly-ubiquitin chains assembled in response to TNF in wild-type (WT) mouse embryonic fibroblasts (MEFs). MEFs were treated with TNF for 15 min and lysed, lysates were subjected to GST PD using GST or GST-NEMO(250-412), beads were treated with buffer or hydroxylamine for 30 min, bound ubiquitin species were then analysed by immunoblotting. (B) Treatment of linear ubiquitin chain assembly complex (LUBAC)-dependent NEMO in vitro ubiquitination reactions with hydroxylamine. All experiments were performed in triplicate representative results are shown.