Figure 7. Overexpression of PLIC-1 decreased Trif abundance in a Nocodazole-sensitive manner. A. 293T cells were transfected with indicated plasmids. 48 hours post-transfection, lysates were prepared and subjected to western blot analysis. HCV NS3/4A was known to cleave Trif and was included as a positive control [8]. However, it only marginally reduced the level of Trif. PLIC-1 has no effect on the expression of a protein named Flap, which was included as a negative control here. B. 293T cells were transfected with 0.2 mg Flag- Trif and indicated amount of HA-PLIC-1 plasmid for 24 hours. Cells were then treated with 30 mM Nocodazole or MG132 (50 mM) for 6 hours prior to cell lysis. Western blotting was performed to quantitate the level of Flag-Trif, HA-PLIC-1. A non-specific band was indicated as the loading control. doi:10.1371/journal.pone.0021153.g007 was obtained from Drs. J. Pomerantz and M. Baldin (Caltech, Pasadena, CA). The p125-luc reporter was provided by Dr. T. Taniguchi (Graduate School of Medicine and Faculty of Medicine, University of Tokyo) and contains the human IFN-b promoter region (2125 to +19) inserted into the luciferase reporter plasmid. The ISRE-Luc plasmid containing 5 copies interferon stimulation PLIC-1 Inhibits TLR3 Activation response element (ISRE) from ISG54 promoter upstream of the firefly luciferase was a gift of Dr. Kui Li (University of Tennessee Health Science Center). YFP-TLR4, Flag-TLR4, HA-PLIC-1, YFP-PLIC-1, Flag-PLIC-1, Fc-TLR4 and pMIR-DFT-empty plasmid, YFP-Trif, Flag-Trif plasmids were constructed in the lab. RFP-LC3 was a gift of Dr. C. Coyne (University of Pittsburgh). GST fusion constructs were obtained from Dr. E. Brown (University of California, San Francisco). pCL-10A1 which is a retrovirus packaging vector was purchased from IMGENEX. shRNA constructs targeting human PLIC-1 (1602, 733) or mouse PLIC-1 (mLMP), as well as the one with the scrambled sequence, were constructed in the lab (detailed information is available upon request). pFlag-MAVS was kindly provided by Dr. J. Chen (UT Southwestern Medical Center, Dallas, TX). Transfection Transfections of 293T and J774 cells were done using lipofectamine 2000 (Invitrogen) reagent following the manufac- turer’s instruction. Briefly, plasmids and media (amount depending on the size of the well) were mixed together, and lipofectamine 2000 (26 of the amount of plasmid) was mixed with media in a separate tube. Then media containing plasmids and media containing lipofectamine were then mixed together, allowed to sit for 15–20 minutes at room temperature, and added slowly to the well. After 4 hours the media was replaced with fresh media containing 5% penicillin and streptomycin, 1% NEAA, and 10% fetal bovine serum. For transfection in J774 macrophage cell line, macrophages were transfected according to AFCS (Alliance for Cellular Signaling gateway) protocol of Transfection of Raw Cells. 16105 cells were plated in each well the day before transfection in 24-well plates. For each transfection, 1 ml Lipofectamine 2000 was added to 25 ml Opti-MEM in a 1.5-ml Eppendorf tube. In another tube the required amount of plasmid was added, mixed, and allowed to sit for 5 min at room temperature. Then, 25 ml DNA mixtures was added to the 25 ml Lipofectamine 2000 mixture and mixed by pipetting. This mixture was incubated for 20 min at room temperature. 200 ml RAWGMI was added to each well and the plasmid/lipofectamine media was then added. After 3 hours the media was removed and replaced with fresh RAWGM1. The composition of RAWGMI media is (for 500 ml) DMEM 435 ml (Final concentration 0.876), FBS 50 ml (Final concentration 10%), HEPES 10 ml (Final concentration 20 mM), L-Glutamine 5 ml (Final concentration 2 mM). Western Blot and Immunoprecipitation For cell lysate preparation, monolayer cells were lysed with a lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P40, 50 mM NaF, 1 mM Na3VO4, 5 mM b-glycero- phosphate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) supplemented with a protease inhibitor mixture (Sigma) on ice. Lysates were cleared by centrifuging at 14,0006 g for 20 min. Boiled samples in 26 SDS loading buffer were resolved on a 10–12% SDS-polyacrylamide gel (SDS-PAGE). After electrophoresis, the separated proteins were transferred onto a nitro-cellulose membrane (Bio-Rad). The resulting blots were blocked in 10% milk for 1 h, and then incubated with primary antibody overnight at 4uC. The secondary antibody used in the immunoblot was a 1:2000 dilution of HRP-linked anti-IgG, followed by detection using the ECL reagents (Amersham). For immunoprecipitation-coupled western blotting, a quantity of 50– 100 mg of total soluble extract was incubated with an appropriate amount of beads conjugated with the specified Ab for 2 h at 4uC. Alternatively, the antibody used for IP was added and kept in a rotating condition for 3–4 hrs and then Protein A beads were PLoS ONE | www.plosone.org 9 June 2011 | Volume 6 | Issue 6 | e21153