mef nucleofectin kit 1  (Amaxa)

Journal: PLoS Pathogens

Article Title: DDX24 Negatively Regulates Cytosolic RNA-Mediated Innate Immune Signaling

doi: 10.1371/journal.ppat.1003721

Figure Lengend Snippet: (A) Schematic of human DDX24 (hDDX24) indicating helicase domains. (B) Schematic of human DDX24 promoter region, indicating STAT1 and IRF7 binding site. (C) Yeast two hybrid assay confirming FADD and DDX24 interactions. (D)293T cells were transiently transfected with c-Myc-DDX24 and FLAG–FADD or control plasmid. Lysates were immunoprecipitated (IP) and immunoblotted (IB) using antibodies to c-Myc or FLAG. (E) On the left, Immunoblot analysis of DDX24 in HUVEC cells treated with DDX24 siRNA or control ns (non-specific) siRNA. On the right, endogenous human DDX24 associates with FADD in HUVEC. Lysates of HUVEC cells were immunoprecipitated with anti-FADD or mouse IgG serum. The immunoprecipitates were analyzed by immunoblot with anti-DDX24 or anti-FADD (top). The expression levels of the endogenous DDX24 and FADD were detected by immunoblot analysis (bottom). (F) Schematic of human DDX24 (hDDX24) indicating helicase domains. 293T cells were transiently transfected with c-Myc-DDX24-FL, c-Myc-DDX24-N or control plasmid with FLAG-FADD, Lysates were immunoprecipitated (IP) using antibodies to FLAG and immunoblotted (IB) using antibodies to c-Myc. (G) Immunoblot analysis of DDX24 in varies human cells or cell lines and MEFs, normalized byGAPDH. (H) MEFs were left untreated or treated with mIFNβat 100 U/ml. Mouse ddx24 and rig-i mRNA were analyzed by RT-PCR. (I) MEFs were left untreated or treated with poly I:C for the indicated time. Mouse ddx24 mRNA was analyzed by RT-PCR. (J) MEFs were left untreated or treated with poly I:C, mIFNβ for the indicated time. Mouse DDX24 and β-actin were detected by immunoblot analysis. (K) HUVEC cells were left untreated or treated with hIFNβ at increasing does. Human DDX24 and β-actin were detected by immunoblot analysis. Data from (H)(I) are presented as means±s.e. from three independent experiments. * indicates P<0.05. ** indicates P<0.01.

Article Snippet: HUVEC and MEFs siRNA transfections were performed using AMAXA HUVEC and MEF Nucleofectin Kit 1 according to the manufacturer's recommendations (AMAXA Biosystems).

Techniques: Binding Assay, Y2H Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: DDX24 Negatively Regulates Cytosolic RNA-Mediated Innate Immune Signaling

doi: 10.1371/journal.ppat.1003721

Figure Lengend Snippet: (A) Loss of mDDX24 affects VSV-luc replication in MEFs. MEFs transfected with ns, mDDX24 or RIG-I siRNA for 72 hours were infected by VSV-luc at M.O.I. = 0.1 or 1. Eight hours and 24 hours post infection, luciferase activities from infected MEF cell lysate were detected. (B) Virus titer from supernatant of (A). (C) Loss of mDDX24 affects VSV-GFP replication in MEFs. Fluorescence microscopy (GFP) of ns, mDDX24 or mRIG-I siRNA treated MEFs following with VSV-GFP infection 24 hours post infection at M.O.I 1. (D) Knockdown efficiency check in MEF. DDX24 and RIG-I antibody are used to detect endogenous DDX24 or RIG-I. (E) Virus titer from supernatant of (C). (F) Loss of hDDX24 affects VSV replication in HUVECs. HUVECs transfected with ns or hDDX24 siRNA for 72 hours were infected by VSV-luc at M.O.I. = 0.1 or 1. Eight hours and 24 hours post infection, plaque assays were performed using supernatants from infected MEFs. HUVEC cell lysates were analyzed by immunoblotting with the indicated antibodies to ensure the knockdown of hDDX24. Data from (A)(B)(E)(F) are presented as means±s.e. from three independent experiments. * indicates P<0.05. ** indicates P<0.01.

Article Snippet: HUVEC and MEFs siRNA transfections were performed using AMAXA HUVEC and MEF Nucleofectin Kit 1 according to the manufacturer's recommendations (AMAXA Biosystems).

Techniques: Transfection, Infection, Luciferase, Fluorescence, Microscopy, Western Blot

Journal: PLoS Pathogens

Article Title: DDX24 Negatively Regulates Cytosolic RNA-Mediated Innate Immune Signaling

doi: 10.1371/journal.ppat.1003721

Figure Lengend Snippet: (A) Schematic of human RIG-I and DDX24. (B) DDX24's binding specificity. 293T cells were transfected with c-Myc-tagged DDX24 for 24 hours before lysed. Pull-down assays were performed by incubating 293T lysate with various biotin-conjugated polynucleotides, and then precipitated with streptavidin beads. Bound proteins were analyzed by immunoblotting with anti-c-Myc antibody. (C) ssRNA transcribed from VSV-G cDNA were conjugated with biotin and utilized in pull-down assays similar to (B). (D) Endogenous DDX24 binds to VSV-G RNA. Pull-down assays were performed by incubating HUVEC cells lysate with biotion-VSV-G. Endogenous DDX24 were analyzed by immunoblotting with anti-DDX24 antibody. (E) DDX24 attenuates RIG-I's RNA binding activity. 293T cells were transfected with c-Myc-tagged DDX24 or FLAG-RIG-I as indicated for 24 hours before lysed. Pull-down assay were performed using biotin-VSV-G and bound proteins were applied to immunoblotting. (F) In vitro translated DDX24 binds to VSV-G RNA. Pull-down assays were performed by incubating in vitro translated DDX24 with biotion-VSV-G or non-labeled VSV-G. DDX24 were analyzed by immunoblotting with anti-c-Myc antibody. (G) In vitro translated DDX24 helicase C domain binds to VSV-G RNA. Pull-down assays were performed by incubating in vitro translated c-Myc-DDX24-N or c-Myc-DDX24-C with biotion-VSV-G or non-labeled VSV-G. DDX24mutants were analyzed by immunoblotting with anti-c-Myc antibody. (H) DDX24 inhibits dRIG-I-, dMDA5, IPS-1 and TBK-1 mediated IFNβ promoter activation. (I)(J) DDX24 blocks synergistical effect of FADD/RIP1 with RIG-I. 293T cells were transfected with reporter plasmid and variant plasmids as indicated. Activations of IFNβ promoter were detected 36 hours post transfection. (K) DDX24 does not affect p53 activation. 293T cells were transfected with reporter plasmid and variant plasmids as indicated. Activations of p53 promoter were detected 36 hours post transfection. Data from (H)(I)(J)(K) are presented as means±s.e. from three independent experiments. * indicates P<0.05. ** indicates P<0.01.

Article Snippet: HUVEC and MEFs siRNA transfections were performed using AMAXA HUVEC and MEF Nucleofectin Kit 1 according to the manufacturer's recommendations (AMAXA Biosystems).

Techniques: Binding Assay, Transfection, Western Blot, RNA Binding Assay, Activity Assay, Pull Down Assay, In Vitro, Labeling, Activation Assay, Plasmid Preparation, Variant Assay