This is different from how SARM regulates NF-κB and IRF3 signaling, which was reported to be mediated by SARM–TRIF interaction. We found that SARM is not only upregulated at the protein level, but also at the mRNA level. Upon LPS challenge, SARM transcription was rapidly upregulated at 1 h and repressed again at 6 h. Furthermore, we provide evidence to suggest that the polybasic

motif and glycine-rich region (GRR) in the N-terminus probably influence the spatial localization and activation of SARM. To investigate the role of SARM and its various domains (Fig. 1A) in MAPK signaling, we first tested SARM’s effect on the activation of AP-1, one of the important transcription factors downstream of TLR signaling. For this purpose, dual luciferase assay for AP-1 was performed in HEK293-TLR4-MD2-CD14 cells. Results showed that SARM significantly inhibited www.selleckchem.com/products/Etopophos.html LPS-stimulated AP-1 activation (Fig. 1B). Truncated SARM containing only the SAM and TIR domains and devoid of the N-terminus (SARMΔN) showed a more potent effect. The TIR domain alone (SARM-TIR) also showed significant inhibition although less potent than the full length SARM and SARMΔN. SARM also inhibited poly (I:C)-mediated AP-1 activation in the HEK293-TLR3 cells (Supporting Information Fig. S2). These results indicate that besides the NF-κB, IRF3 and IRF7 inhibition 23, SARM also inhibits

the activity of AP-1. Interestingly, transfection of equimolar amounts of the three constructs resulted in markedly different levels of protein expression, with the SARM-TIR extremely high, SARMΔN at detectable level and the full-length SARM very low (or even undetectable, Supporting Information Fig. S3). Dactolisib in vitro However, this may be attributable to different qualities of the plasmid preparations and may not necessarily reflect a biological reason, although the A260/A280 values and the amounts of circular plasmids for each construct were comparable. Although SARM-TIR appears more expression Etomidate competent than SARMΔN, its functional effect is the lowest. This suggests that the SAM domain, which is present in SARMΔN but absent in SARM-TIR, plays an important role in its inhibition

function, which is consistent with a previous report 23. Moreover, the higher expression of SARMΔN compared to the full-length SARM might contribute to the greater effect of SARMΔN. Thus, at this juncture, we cannot ascertain that SARMΔN protein is more potent than the full-length SARM protein. Nevertheless, the presence of the N-terminus and SAM domains seems to reduce the expression and/or stability of the protein. This might be a strategy to control the SARM activity in vivo so as to avoid detrimental effects to the host. To investigate whether the AP-1 inhibition is specific to the TRIF-mediated pathway, we transfected HEK293 cells with AP-1 reporter and TRIF- or MyD88-expressing plasmid, with or without one of the three SARM constructs: full-length SARM, SARMΔN or SARM-TIR (Fig. 1A).