Fig 5

(A) TMEM33 interacts with MAVS, MITA, TBK1, and IRF3. EPC cells seeded in 10 cm2 dishes were transfected with the indicated plasmids (5 ?g each). After 24 h, cell lysates were immunoprecipitated (IP) with anti-Flag affinity gel. Then the immunoprecipitates and WCLs were analyzed by IB with the anti-Myc and anti-Flag Abs, respectively. (B and C) TMEM33 is localized at the ER. EPC cells seeded onto microscopy cover glass in 6-well plates were co-transfected with 1 ?g TMEM33-EGFP and 1 ?g empty vector (B) or ER-DsRed (C). After 24 h, the cells were fixed and subjected to confocal microscopy analysis. Green signals represent overexpressed TMEM33, and blue staining indicates the nucleus region. The yellow staining in the merged image indicates that TMEM33 is localized at the ER. (D-G) TMEM33 colocalizes with MAVS and MITA. EPC cells were plated onto coverslips in 6-well plates and co-transfected with 1 ?g TMEM33-EGFP and 1 ?g MAVS-DsRed (D), TBK1-DsRed (E), MITA-DsRed (F), or IRF3-DsRed (G). After 24 h, the cells were fixed and observed by confocal microscopy. Green signals represent overexpressed TMEM33. Red signals represent overexpressed MAVS, TBK1, MITA, or IRF3, and blue staining indicates the nucleus region. The yellow staining in the merged image indicates the colocalization of TMEM33 and MAVS or MITA (original magnification 63×; oil immersion objective). Scale bar, 10 ?m. (H) The colocalization rates of B-G were performed by LAS AF Lite. All experiments were repeated for at least three times with similar results.