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. 2018 May 7;128(6):2459–2472. doi: 10.1172/JCI95720

Figure 3. Phosphorylation of sNASP regulates its interaction with TRAF6 and affects cytokine production.

Figure 3

(A) Raw264.7 cells were transfected with GFP-tagged sNASP, stimulated with LPS, and assessed by IB with antibody against phosphorylated serine or GFP after IP with anti-GFP or by IB with anti-GFP in TCL. (B) Phosphorylation of the serine residue of endogenous sNASP in THP-1 cells following LPS stimulation, assessed by IB with antibody against phosphorylated serine (pSerine) or NASP after IP with anti-NASP. TCL IB was done with anti-TRAF6. (C) THP-1 cells were transfected with GFP-tagged WT sNASP or S158A, S164A, S158E mutants, followed by IB with antibody against phosphorylated serine, TRAF6, or GFP after IP with anti-GFP. TCL IB was done with anti-TRAF6 or anti–β-actin (below). (D) THP-1 cells were transfected with GFP-tagged WT sNASP or S158A, S158E mutants, followed by IB with antibody against Ub, TRAF6, or NASP after IP with anti-TRAF6. TCL IB was done with anti-TRAF6, anti-GFP, anti-pTAK1, anti-TAK1, or anti–β-actin. (E) Expression of TNF-α and IL-6 in Raw264.7 cell lines transfected with WT sNASP, S158A, S158E mutants, or EV and stimulated with LPS. Results were normalized to the expression of ACTB (encoding β-actin) and untreated cells. (F) Secretion of TNF-α and IL-6 by Raw264.7 cells transduced as in E and stimulated with LPS. Data are mean ± SE for each group. A–F represent a minimum of 3 independent experiments.