Page 105 - AN-4-1

Advanced Neurology TDP-43 regulates IFN1 production

A B C

Figure 2. TDP43 reduces TBK1-induced IRF7 expression. (A) Immunoblot analysis of TBK1, IRF3, and IRF7 protein levels in HEK-293T cells transfected
with TDP43 and TBK1. Each lane was loaded by 10 µg of total protein, and GAPDH was served as a loading control. Proteins were detected using Myc and
FLAG tags. (B) Densitometric analysis of p-TBK1, Myc-tagged TBK1, p-IRF3, IRF3, and IRF7 protein levels based on data from panel A. (C) Immunoblot
analysis of Myc, TBK1, p-TBK1, p-IRF3, IRF3, and IRF7 protein levels in HEK-293-T cells co-transfected with TBK1 (2 µg) and different concentrations
of TDP43 (0, 0.2, 0.5, and 1.5 µg). The data are shown as mean ± standard deviation (n = 5 per group). ***P < 0.001.
Abbreviations: ns: Not significant; TBK1: TANK-binding kinase 1; p-TBK1: phosphorylated-TANK-binding kinase 1; TDP43: TAR DNA-binding protein
43; IRF3: Interferon regulatory factor 3; p-IRF3: phosphorylated-interferon regulatory factor 3; IRF7: Interferon regulatory factor 7.
A
A

B C
B
C

Figure 3. IRF7 overexpression alleviates TDP43-mediated IFN1 suppression. (A) Quantitative PCR analysis of IRF3, IRF7, IFN1, and ISGs (ISG54 and
ISG56) in HEK-293T cells co-expressing TBK1 and TDP43, along with IRF3 or IRF7. (B) Immunoblot analysis of p-TBK1, Myc-TBK1, p-IRF3, IRF3,
and IRF7 protein levels. (C) IFN-β activity measured by dual-luciferase reporter assay. HEK-293T cells were co-transfected with luciferase-tagged IFN-β,
Renilla-tagged TK reporter, and plasmids encoding TBK1 and TDP43, along with increasing concentrations of IRF7 (0, 0.2, 0.5, and 1.5 µg). The assay was
conducted at 24 h post-transfection. The data are shown as mean ± standard deviation (n = 5 per group). *P < 0.05, **P < 0.01, and ***P < 0.001.
Abbreviations: TBK1: TANK-binding kinase 1; TDP43: TAR DNA-binding protein 43; IRF3: Interferon regulatory factor 3; IRF7: Interferon regulatory
factor 7; IFNA: Interferon-alpha; IFNB: Interferon-beta; ISG54: Interferon-stimulated gene 54; ISG56: Interferon-stimulated gene 56; IL6: Interleukin-6;
IL8: Interleukin-8; p-TBK1: phosphorylated-TANK-binding kinase 1; p-IRF3: phosphorylated-interferon regulatory factor 3.

Volume 4 Issue 1 (2025) 99 doi: 10.36922/an.6272