Fig 9

(A) The amount of TBK1-phosphorylated TMEM33 is reduced by CIP treatment. EPC cells were seeded in 6-well plates overnight and transfected with the indicated plasmids (1 μg each) for 24 h. The cell lysates (100 μg) were treated with or without CIP (10 U) for 40 min at 37°C. Then the lysates were detected by IB with the indicated Abs. (B and C) TBK1 mediates the phosphorylation of MITA (B) and IRF3 (C). EPC cells were seeded into 6-well plates overnight and transfected with the indicated plasmids (1 μg each) for 24 h. The cell lysates (100 μg) were treated with or without CIP (10 U) for 40 min at 37°C. The lysates were then subjected to IB with the indicated Abs. (D and E) Overexpression of TMEM33 inhibits TBK1-mediated phosphorylation of MITA (D) and IRF3 (E) in a dose-dependent manner. EPC cells were seeded into 6-well plates overnight and co-transfected with 1 μg TBK1-Flag and 1 μg empty vector or TMEM33-Myc (0.5 and 1 μg, respectively), together with 1 μg MITA-HA or IRF3-HA for 24 h. The lysates were then subjected to IB with the indicated Abs. (F) TBK1 phosphorylates wild-type TMEM33 and TMEM33-ΔTM3. EPC cells were seeded in 6-well plates overnight and transfected with the indicated plasmids (1 μg each) for 24 h. Then the lysates were detected by IB with the indicated Abs. (G and H) TMEM33-ΔTM1 and TMEM33-ΔTM2 have no effect on TBK1-mediated phosphorylation of MITA (G) and IRF3 (H). EPC cells were seeded into 6-well plates overnight and co-transfected with 1 μg TBK1-Flag and 1 μg empty vector, TMEM33-Myc, TMEM33-ΔTM1-Myc, TMEM33-ΔTM2-Myc, or TMEM33-ΔTM3-Myc together with 1 μg MITA-HA or IRF3-HA for 24 h. The lysates were then subjected to IB with the indicated Abs. (I and J) TMEM33 blocks the interaction between TBK1 and MITA in a dose-dependent manner. EPC cells seeded in 10 cm² dishes were co-transfected with 4 μg TBK1-Flag and 4 μg MITA-HA (I) or together with TMEM33-Myc (2 or 4 μg) (J). After 24 h, cell lysates were immunoprecipitated (IP) with anti-Flag affinity gel. Then the immunoprecipitates and WCLs were analyzed by IB with the indicated Abs. (J and K) TMEM33 disrupts the interaction between TBK1 and IRF3 in a dose-dependent manner. EPC cells seeded in 10 cm² dishes were co-transfected with 4 μg TBK1-Flag and 4 μg IRF3-Myc (J) or together with TMEM33-HA (2 or 4 μg) (K) for 24 h. Immunoprecipitation and immunoblot analysis were performed similarly as in I and J. All experiments were repeated for at least three times with similar results.