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. 2017 Oct 9;114(43):11392–11397. doi: 10.1073/pnas.1705748114

Fig. S3.

Fig. S3.

Development of a functional assay for CARD9 ubiquitinylation by TRIM62. (A and B) AlphaLISA assay detects TRIM62-mediated CARD9 ubiquitinylation in the presence of WT TRIM62 and WT CARD9. Ubiquitinylation of CARD9 was significantly decreased with TRIM62 C11A (ligase-dead mutant), CARD9Δ11, or CARD9 K125R lacking a key lysine for TRIM62-mediated ubiquitinylation. Data are expressed as mean AlphaLISA signal ± SD. (C) The family of hit compounds tested in the Truhit AlphaLISA assay designed for detection of nonspecific AlphaLISA inhibitors. Data are expressed as mean of percent AlphaLISA signal (100% = mean AlphaLISA signal with DMSO control) ± SD. (D) Western blotting detects the inhibition of in vitro CARD9 ubiquitinylation reaction by the family of the hit compounds. Results are representative of three independent experiments.