St that obesity-induced inflammation leads to dysfunction of brown adipocytes by means of the reduction of UCP1 and other thermogenic markers. Nonetheless, the regulatory mechanisms of inflammation in brown adipocytes remain largely obscure. The NOD-RIPK2 Brain Derived Neurotrophic Factor (BDNF) Proteins Storage & Stability pathway plays a vital part in host defense against bacterial infection and is related together with the onset of autoimmune disorders9. In a cell beneath bacterial infection, intracellular pattern recognition receptors sense the peptidoglycan derivatives of bacterial cell wall; which is, nucleotide-binding oligomerization domain 1 (NOD1) and NOD2 recognize meso-diaminopimelic acid (DAP) and muramyl dipeptide (MDP), respectively. Upon ligand binding, NODs oligomerize through the caspase recruitment domain (CARD) and induce further oligomerization of another CARD-containing protein, receptor-interacting serine/threonineprotein kinase 2 (RIPK2). Oligomerized RIPK2 is K63-polyubiquitinated by X-linked inhibitor of apoptosis protein (XIAP), linear ubiquitin chain assembly complex (LUBAC), as well as other E3 ligases and additional CCL22 Proteins Molecular Weight recruits its downstream effectors, like TGF-beta activated kinase 1 (TAK1)/TAK1 binding protein (TAB) complex and nuclear aspect of kappa B (NF-B) crucial modulator (NEMO) complicated. Consequently, the c-jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and NF-B pathways are activated, leading for the induction of proinflammatory cytokines10. Along with the function in immune cells, the NOD-RIPK2 pathway is implicated in adipose inflammation and affects the physiology of adipocytes. In adipocytes, pattern recognition receptors such as NOD1 are regarded to become activated by bacterial fragments translocated from gut microbiota11, that is augmented under obesity12. NOD1 activation in white adipocytes induces insulin resistance and lipolysis135 and suppresses adipocyte differentiation with attenuated expression of adipocyte markers and lipid accumulation16. Moreover, NOD1 activation in brown adipocytes leads to suppression of brown adipocyte markers, including UCP117. These lines of evidence recommend that the inflammatory NOD-RIPK2 pathway in adipocytes suppresses the differentiation of adipocytes. We have previously reported apoptosis signal-regulating kinase 1 (ASK1)18 as a critical regulator of thermogenesis; below -adrenergic receptor stimulation, protein kinase A (PKA) activates the ASK1-p38 MAPK axis to induce brown adipocyte-specific genes19,20. Here, we show that ASK1 suppresses the NOD-RIPK2 pathway in brown adipocytes. We report an analog sensitive kinase allele (ASKA) technology-based pull-down mass spectrometry (MS) method and identify RIPK2 as a novel interactor of ASK1 in brown adipocytes. ASK1 interferes with the NOD-RIPK2 pathway by inhibiting the activation from the RIPK2 signaling complex. As a possible biological significance, our in vitro model for intercellular thermogenic regulation implies that the suppressive function of ASK1 in the NOD-RIPK2 pathway positively contributes towards the upkeep of thermogenic function in BAT under inflammation, which suggests a complementary role towards the ASK1’s function as a positive regulator of BAT thermogenesis via PKA-ASK1-p38 axis. This function demonstrates an instance application of our novel chemical pull-down technique and reveals the multifaceted finetuning role of ASK1 in brown adipocytes.Resultsnisms or functions of ASK1 in BAT, we initially sought to recognize components on the ASK1 signalosome in brown adipocyte.