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Fig. 4 TMEM47 interacts with the MAVS/STING and colocalizes with STING at the ER. (A) TMEM47 associates with the MAVS and STING. EPC cells seeded in 10-cm2 dishes were transfected with the indicated plasmids (5 µg each). After 24 h, cell lysates were immunoprecipitated (IP) with anti-Flag affinity gel. Then the immunoprecipitates and whole cell lysates (WCLs) were analyzed by IB with anti-Flag and anti-Myc Abs, respectively. (B and C) TMEM47 colocalizes with STING at the ER. EPC cells were plated onto coverslips in six-well plates and co-transfected with 1 µg TMEM47-EGFP and 1 µg empty vector, MAVS-DsRed/STING-DsRed (B), or ER-DsRed (C), respectively. After 24 h, the cells were fixed and subjected to confocal microscopy analysis. Green signals represent overexpressed TMEM47, red signals represent overexpressed MAVS, STING, or ER, and blue staining indicates the nucleus region (original magnification 63×; oil immersion objective). Scale bar, 5 µm. (D and F) Schematic representation of full-length MAVS/STING and deletion mutants in this study. (E and G) TMEM47 interacts with MAVS via its PRO domain and associates with STING by its TM domain. EPC cells seeded in 10-cm2 dishes were transfected with the indicated plasmids (5 µg each). After 24 h, cell lysates were IP with anti-Myc (E) or anti-Flag (G) affinity gel. Then the immunoprecipitates and WCLs were analyzed by IB with anti-Myc, anti-HA, and anti-Flag Abs, respectively. All experiments were repeated for at least three times with similar results.