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. 2021 May 11;12:2713. doi: 10.1038/s41467-021-22979-3

Fig. 2. IL-1β is modified by K48-linked, K63-linked, and K11-linked ubiquitin chains and is targeted for proteasomal destruction.

Fig. 2

a BMDMs were stimulated with Lipopolysaccharide (LPS, 100 ng/ml) for 3 h and Q-VD-OPh (20 μM) added in the last 30 min of priming. Cells were then treated with the protein synthesis inhibitor cycloheximide (CHX, 20 μg/ml) for up to 6 h, as indicated, and total cell lysates analyzed for the indicated proteins by immunoblot. One of two experiments. b BMDMs were stimulated with LPS (100 ng/ml) for 2.5 h; with Q-VD-OPh (20 μM) added in the last 30 min alongside, as indicated, the proteasome inhibitor MG132 (20 μM) and the inhibitor of lysosome function bafilomycin A1 (BafA1 300 nM). Cells were then treated with cycloheximide (CHX, 20 μg/ml) for up to 6 h as specified. Total cell lysates were subjected to immunoblot for the indicated proteins. One of three experiments. c BMDMs were treated with LPS (50 ng/ml) for up to 8 h, and MG132 (20 μM) added for the specified times. Ubiquitylated proteins were isolated from cell lysates using Tandem Ubiquitin Binding Entity (TUBE) purification, and cell lysate input and TUBE purified ubiquitylated (Ub.) proteins analyzed by immunoblot. One of three experiments. d iBMDMs were treated with LPS for 6 h and ubiquitylated proteins isolated by Glutathione S-transferase-Ubiquitin Associated domain (GST-UBA) purification. Specific ubiquitin chain linkages were cleaved via treatment with the indicated DUBs: USP21 (total ubiquitin), AMSH (K63-linked), OTUB1 (K48-linked), vOTU (total ubiquitin except M1), 200 and 500 nM Cezanne (Cez., K11-linked) and OTULIN (M1 linear), and immunoblots performed for the indicated proteins. One of three experiments.