TRIM31 and NLRP3 Protein Binding Assay

NLRP3, TRIM31 WT and ΔRING mutant proteins were expressed with a TNT Quick Coupled Transcription/Translation System (Promega) according to the instructions of the manufacturer22 (link). Binding assays were performed by mixing Flag-tagged TRIM31 and NLRP3 together, followed by IP with Flag antibody and WB with NLRP3 antibody. Ubiquitination was analysed with an ubiquitination kit (Boston Biochem) following protocols recommended by the manufacturer.

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Song H., Liu B., Huai W., Yu Z., Wang W., Zhao J., Han L., Jiang G., Zhang L., Gao C, & Zhao W. (2016). The E3 ubiquitin ligase TRIM31 attenuates NLRP3 inflammasome activation by promoting proteasomal degradation of NLRP3. Nature Communications, 7, 13727.

Publication 2016

TNT Quick Coupled Transcription/Translation System ubiquitination kit

Antibody Assays Mutant proteins Promega Transcription Ubiquitination

Corresponding Organization :

Other organizations : Shandong University, Shandong First Medical University

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Variable analysis

independent variables

TRIM31 WT and ΔRING mutant proteins

dependent variables

Binding between Flag-tagged TRIM31 and NLRP3
Ubiquitination of proteins

control variables

Experimental protocols and conditions followed as per manufacturer's instructions

controls

Positive control: Binding of Flag-tagged TRIM31 and NLRP3 (as measured by IP and WB)
Negative control: Not specified

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