Fig. 4

Tet2 interacts with Stat1 in the cytoplasm of macrophages and suppresses Stat1 phosphorylation during IFN-γ signaling activation. A, B Macrophages were stimulated with 10 ng/mL IFN-γ for the indicated times (A) or with the indicated IFN-γ concentrations for 24 h (B). Tet2, Stat1, p-Stat1 (Tyr701), and the GAPDH protein level were detected using western blotting. The protein was normalized to GAPDH expression levels. Tet2: ten-eleven translocation- 2, Stat1: signal transducer and activator of transcription 1, p-Stat1: phosphor-Stat1, GAPDH: glyceraldehyde-3-phosphate dehydrogenase. A Macrophages were treated with Tet2 inhibitor-BC339 or DMSO as a control for 24 h and stimulated with 10 ng/mL IFN-γ for the indicated time. Tet2, Stat1, p-Stat1 (Tyr701), and GAPDH protein levels were detected using western blotting. The protein was normalized to GAPDH expression levels in each sample. B Macrophages were treated with Tet2 inhibitor-BC339 or DMSO as a control for 24 h and stimulated with 10 ng/mL IFN-γ for 12 h. The mRNA expression levels of Tet2, Irf1, Ifit1, and Cxcl10 were examined through qPCR analysis. The genes were normalized to GAPDH mRNA levels in each sample. Irf1: interferon regulatory factor 1, Ifit1: interferon-induced protein with tetratricopeptide repeats 1, Cxcl10: C-X-C motif chemokine ligand 10. C Co-immunoprecipitation (co-IP) assays were performed using an anti-Tet2 antibody and IgG as a control. Tet2 and Stat1 protein levels were detected using western blotting. D Macrophages were fixed and incubated with an anti-Tet2 antibody (red), anti-Stat1 antibody (green), and DAPI (blue) and visualized using confocal microscopy. E Macrophages were treated with Tet2 inhibitor-BC339 or DMSO as a control for 24 h and stimulated with 10 ng/mL IFN-γ for the indicated time. Co-IP assays were performed using an anti-Stat1 antibody and IgG as control. Tet2, Stat1, Jak1, and Jak2 protein levels were detected using western blotting. (Jak1: Janus kinase 1, Jak2: Janus kinase 2, A to G, n = 3 *p < 0.05)