Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2020 Jan 30;217(4):e20182091. doi: 10.1084/jem.20182091

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2020 Tang et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 5. — Identification of RNF125 as an additional E3 ubiquitin ligase that initiates K63-linked polyubiquitination of NLRP3. (A) Immunoblot analysis of NLRP3 immunoprecipitates (IP) and total cell lysates (CL) from WT BMDMs primed with LPS and stimulated with ATP (2.5 mM) for 5 and 15 min with antibodies against TRIM14, TRIM21, TRIM31, TRIM47, RNF125, RNF213, CBLB, and NLRP3. (B) IL-1β production by BMDMs from WT mice (n = 3) that were transfected with siRNAs specific for Trim14, Trim21, Trim31, Trim47, Rnf125, and Rnf213, primed with LPS, and stimulated with ATP. Data are represented as mean ± SD.P < 0.001 (Rnf125 siRNA-transfected BMDMs vs. other siRNA-transfected BMDMs); Student's t test. Cell lysates from the above BMDMs were blotted with antibodies against TRIM14, TRIM21, TRIM31, TRIM47, RNF125, RNF213, and actin. Ctr, control. (C) Anti-His immunoblot analysis of Flag immunoprecipitates of lysates from Rnf125 gene-silenced HEK293T cells transfected with Flag-tagged NLRP3 or NLRP3 LRR (K>R), Myc-tagged RNF125, or Myc-RNF125 RM mutant together with His-tagged K63 ubiquitin. (D) Anti-K63 ubiquitin immunoblot analysis of NLRP3 immunoprecipitates of lysates from WT BMDMs transfected with Rnf125 siRNA or a control siRNA, primed with LPS, and stimulated with ATP. (E) Anti-K63 and K48 immunoblot analysis of Flag immunoprecipitates of lysates from HEK293T cells transfected with Flag-tagged NLRP3, HA-tagged Cbl-b, Myc-RNF125, and His-tagged ubiquitin treated with or without AMSH. (F) Anti-Myc immunoblot analysis of Flag immunoprecipitates of lysates from HEK293T cells transfected with Myc-tagged RNF125, RNF125 (1–76), RNF125 (Δ1-76), or RNF126 (ZF), together with Flag-tagged NLRP3. (G) Anti-HA immunoblot analysis of Flag immunoprecipitates of lysates from HEK293T cells transfected with Flag-tagged NLRP3 LRR or NLRP3 LRR (K/R) mutant, HA-tagged Cbl-b UBA, Myc-RNF125 or RNF125 RM, and His-tagged K63 ubiquitin in the presence of MG132 (10 µM, overnight). (H) Anti–K48-ubiquitin immunoblot of Flag immunoprecipitates of lysates from HEK293T cells transfected with Flag-tagged NLRP3 or NLRP3 LRR (K/R) mutant, HA-tagged Cbl-b, Myc-RNF125, and His-tagged K63 ubiquitin. (I) Anti-RNF125 immunoblot analysis of NLRP3 immunoprecipitates of lysates from WT and Cblb^−/− BMDMs that were primed with LPS and stimulated with ATP. (J) Anti-NLRP3 immunoblot analysis of Cbl-b immunoprecipitates of lysates from WT BMDMs transfected with Rnf125 siRNA or a control siRNA, primed with LPS, and stimulated with ATP. (K) Anti-NLRP3 and anti-RNF125 immunoblot analysis of lysates from WT BMDMs transfected with Rnf125 siRNA or a control siRNA, primed with LPS, and stimulated with ATP. (L) Anti-NLRP3 and anti–pro-IL-1β immunoblot analysis of WT BMDMs transfected with Rnf125 siRNA or a control siRNA, primed with LPS for 1, 2, and 4 h. Data are representative of three independent experiments. Actin was used as a loading control.