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. 2012 Dec 20;8(12):e1003086. doi: 10.1371/journal.ppat.1003086

Figure 6. COX5B coordinates with ATG5 to regulate MAVS signaling.

Figure 6

(A) Hela cells were transfected with LC3-GFP (Green) and control vector or Flag-MAVS (Red) for 24 h. Cells were fixed, then stained with anti-Flag antibody, and imaged by confocal microscopy. (B) Hela cells were transfected with increasing amounts of MAVS expression plasmids, and empty vector was used to balance the total DNA amount. Total protein was prepared and subjected to immunoblot analysis after 36 h transfection. (C) HEK293 cells were transfected with increasing amounts of MAVS expression plasmids, and empty vector was used to balance the total DNA amount. Twenty-four hours after transfection, total protein was extracted and subjected to immunoblot analysis. (D) Cell transfection described as seen in (C), after transfection, the total RNAs were prepared for real-time PCR analysis. (E) HEK293 cells were transfected as indicated expression plasmids. Twenty-four hours after transfection, cell lysates were prepared, immunoprecipitated with anti-Flag beads, followed by immunoblot analysis with the indicated antibodies. (F) HEK293 cells were transfected COX5B oligo or NC, after 30 h, transfected again with MAVS or empty vector plasmids, 24 h later, cells were lysed for western blotting. (G) ATG5 and COX5B RNAi oligos (final concentration 20 nM) were transfected into HEK293 cells, NC oligos were used to balance the equal amount of RNAi oligos. Thirty-six hours after transfection, cells were transfected again with the expression plasmids for MAVS or empty vector along with the reporter plasmids followed by measurement of luciferase assays after 24 h transfection. (H) ATG5 and COX5B RNAi oligo transfection was performed as described in (G), and then cells were transfected again with reporter plasmids. Eight hours after second transfection, cells were infected with Sendai virus for 16 h followed by measurement of luciferase assays. Data from D, G and H are representative of at least three independent experiments (mean and s.d. of duplicate assays). *, P<0.05; **, P<0.01 versus control groups.