Rabbit Anti-Human RNF144B (a.a. 218-303) Polyclonal antibody

Differences in human and mouse immune responses may partly reflect species-specific adaptations and can provide important insights into human immunity. In this study, we show that RNF144B, which encodes an E3 ubiquitin ligase, was lipopolysaccharide-inducible in primary human macrophages and in human macrophage-like THP-1 cells. In contrast, Rnf144b was not lipopolysaccharide-inducible in several mouse cell populations, including primary macrophages from C57BL/6 and BALB/c mice and RAW264.7 macrophages. Similarly, Rnf144b was not up-regulated by infection of C57BL/6 mice with Escherichia coli. Although the human and mouse RNF144B genes have conserved transcription start sites, cap analysis of gene expression data confirmed that the RNF144B promoter directs transcription in human but not mouse macrophages. The human andmouse RNF144B genes are controlled by highly conserved TATA-containing promoters, but subtle differences in transcription factor binding sites may account for differential regulation. Using gene silencing, we showed that RNF144B is necessary for priming of inflammasome responses in primary human macrophages. Specifically, RNF144B promotes lipopolysaccharide-inducible IL-1b mRNA expression but does not regulate expression of several other lipopolysaccharide-inducible cytokines (e.g., interleukin-10, interferon-gamma) or affect expression of inflammasome components or substrates (e.g., procaspase-1, pro-interleukin-18). Our findings thus revealed a species-specific regulatory mechanism for selective inflammasome priming in human macrophages.

Several ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage- specific "affimer'' reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer. Structure-guided improvements yield superior affinity reagents suitable for western blotting, confocal fluorescence microscopy and pull-down applications. This allowed us to identify RNF144A and RNF144B as E3 ligases that assemble K6-, K11-, and K48-linked polyubiquitin in vitro. A protocol to enrich K6-ubiquitinated proteins from cells identifies HUWE1 as a main E3 ligase for this chain type, and we show that mitofusin-2 is modified with K6-linked polyubiquitin in a HUWE1-dependent manner.