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

Figure 7. Heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L) generates ester linkages on heterotypic chains but requires HOIL-1-interacting protein (HOIP) catalytic activity to polymerize ubiquitin.

(A) Comparison of linear ubiquitin chain assembly complex (LUBAC) in vitro chain assembly by complexes containing different catalytically inert mutants of HOIP and HOIL-1L. (B) OTULIN restriction of poly-ubiquitin chains assembled by LUBAC containing wild-type (WT) or catalytically inert HOIL-1L. (C) Hydroxylamine treatment of M1 linkage-containing poly-ubiquitin chains assembled in response to TNF in WT and Rbck1C458A/C458A mouse embryonic fibroblast (MEF) cells. Cells were treated with TNF (50 ng/ml) for 15 min and lysed, lysates were subjected to GST PD using GST-NEMO (250-412), beads were treated with buffer or hydroxylamine for 30 min, bound ubiquitin species were then analysed by immunoblotting. (D) Schematic representations of different HOIL-1L mutants. (E) Comparison of LUBAC in vitro chain assembly by complexes containing different HOIL-1L mutants. All experiments were performed in triplicate representative results are shown.

Figure 7.

Figure 7—figure supplement 1. Purification of linear ubiquitin chain assembly complex (LUBAC) containing catalytically inert HOIL-1-interacting protein (HOIP) and heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L) proteins.

Figure 7—figure supplement 1.

(A) SDS-PAGE analysis of different purified LUBAC. (B) Immunoblot analysis of different purified LUBAC.
Figure 7—figure supplement 2. Generation of Hoil-1lC458A/C458A mice.

Figure 7—figure supplement 2.

(A) Sequences of genomic DNA around C458 codon, gRNAs, and donor oligonucleotide used to target heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L) C458A mutation. (B) Sanger sequencing confirming correct mutations at target sites. (C) Genotyping results of Hoil-1l+/+, Hoil-1l+/C458A, and Hoil-1lC458A/C458A mice. Hpy188III digest of a PCR fragment confirming correct targeting where a silent mutation is inserted. (D) Immunoblot analysis of linear ubiquitin chain assembly complex (LUBAC) component expression in mouse embryonic fibroblasts (MEFs) derived from Hoil-1l+/+ and Hoil-1lC458A/C458A mice.