Figure 5. REG3A activates EXTL3-AKT-STAT3 to induce SHP-1.

(a) Interaction between Flag-tagged different domains of EXTL3 and REG3A assessed by immunoblot analysis after immunoprecipitation with anti-Flag or anti-REG3A. M: mock; F: full-length EXTL3; N: N-EXTL3 (1–548); C: C-EXTL3 (141–919); ΔNΔC: EXTL3 ΔNΔC (141–548). (b) Interaction between REG3A or C-REG3A and Flag-tagged N-EXTL3 assessed by immunoblot analysis after immunoprecipitation with anti-REG3A or anti-Flag. F: full-length REG3A; C: CTLD domain of REG3A. (c) Quantification of TNF-α and IL-6 mRNA expression in NHEKs stimulated with 5 μg ml⁻¹ poly(I:C) and/or 30 nM REG3A before or after EXTL3 silencing (n=3). (d) Immunoblot of SHP-1 in NHEKs treated with 30 nM REG3A before or after EXTL3 silencing. EXTK3 si: EXTL3 siRNA. (e) SHP-1 production in NHEKs treated with 30 nM REG3A in the presence or absence of NF-κB inhibitor (Bay11, 10 μM), Erk inhibitor (PD98059, 20 μM), p38 MAPK inhibitor (SB202190, 5 μM), PI3K inhibitor (LY294002, 50 μM), STAT3 inhibitor (S3I-201,50 μM) and AKT inhibitor (AKT1/2 inhibitor, 8 μM). (f) Immunoblot of phosphorylated AKT in NHEKs treated with 30 nM REG3A for 1 h before or after EXTL3 was silenced. (g) Immunoblot of p-STAT3 and p-AKT in NHEKs treated with 30 nM REG3A and/or AKT1/2 inhibitor for 1 h. AKT1/2 i: AKT1/2 inhibitor. (h) Immunoblot of p-STAT3 and p-AKT in NHEKs treated with 30 nM REG3A and/or STAT3 inhibitor (S3I-201) for 1 h. STAT3 i: STAT3 inhibitor. ***P<0.001. NS, no significance. P values were analysed by two-way analysis of variance (ANOVA). Data are means±s.e.m. and representative of two to three independent experiments.