Figure 1.

NOD2/TLR2-mediated phosphorylation of ataxin-3. Immunoblot using antibodies against ataxin-3 and β-actin of whole cell lysates (WCL) and phosphoprotein enriched lysates (PE) from moDCs either (A) unstimulated or stimulated with the NOD2 ligand MDP 10 μg/ml, or the TLR2 ligand PAM₃CSK₄ 1 μg/ml, or both, or the TLR4 ligand LPS 100 ng/ml or the TLR7/8 ligand R848 (Resiquimod) 1 μg/ml for 30 min or (B) unstimulated or stimulated with the NOD2 ligand MDP for 15, 30, or 60 min. (C) THP-1 cells were transduced with control or NOD2-targeting lentiviral shRNAs and analyzed for NOD2 expression by qPCR analysis. (D) Immunoblot using antibodies against ataxin-3, p38, and β-actin of WCL and PE lysates from THP-1 cells expressing control or NOD2 shRNA and either unstimulated or stimulated with the NOD2 ligand MDP or the TLR ligand PAM₃CSK₄, or both, for 60 min. (E) Immunoblot using antibodies against ataxin-3, p38 and β-actin of WCL and PE lysates from THP1 cells which were pre-treated with the RIPK2 inhibitor Ponatinib 50 nM for 60 min and then left unstimulated or stimulated with the NOD2 ligand MDP or the TLR2 ligand PAM₃CSK₄ or both. (F) THP-1 cells were transduced with control or TBK1-targeting lentiviral shRNAs and immunoblotted to detect TBK1 expression (G) Immunoblot using antibodies against ataxin-3, p38 and b-actin of WCL and PE lysates from THP-1 cells expressing control or TBK1 shRNA left unstimulated or stimulated with the NOD2 ligand MDP or the TLR2 ligand PAM₃CSK₄ or both for 60 min. (H) Immunoblot using antibodies against TBK1 and ataxin-3 following an in vitro kinase assay of GST-TBK1 protein or His-ataxin-3 protein with ATP, or both GST-TBK1 and His-ataxin-3 with or without ATP which were incubated for 60 min at 30°C. (I,J) Identification of the phosphorylated serine residue (s265) in ataxin-3 with a characteristic increase in mass/charge ratio in the stimulated sample. All immunoblots are representative of at least two independent experiments.