fluorescence module of glomax-multi microplate multimode reader  (Promega)

Figure Legend Snippet: ( a ) Immunoblot analysis of MARCH5 expression levels in MARCH5-depleted Raw264.7 cells. See full blots in . ( b ) Twenty-four hours after infection, PR8-GFP or VSV-GFP virus replication assay by fluorescence microscopy (fluorescence, upper; phase-contrast microscopy, bottom) and virus titration by fluorescence analysis or plaque assay in siControl or siMARCH5 expressing Raw264.7 cells. Error bars, mean±s.e.m. ( n =3). Scale bars, 100 μm. ( c ) Determination of the virus titration using fluorescence analysis or plaque assay in March5 +/+ and March5 +/− BMDM cells infected with PR8-GFP or VSV-GFP virus. Error bars, mean±s.e.m. ( n =3). ( d , e ) Bioassay of IFN-β or IL-6 (ELISA) in supernatants of MARCH5-depleted Raw264.7 ( d ) or March5 +/+ and March5 +/− BMDM cells ( e ) infected or transfected with PR8-GFP, VSV-GFP, poly(I:C) or 5′ppp-dsRNA. After cells were transfected with siControl or siMARCH5 for 24 h, followed by infection with virus or transfection with poly(I:C) for indicated times. Error bars, mean±s.e.m. ( n =3). All data are representative of at least three independent experiments.

Techniques Used: Western Blot, Expressing, Infection, Virus, Fluorescence, Microscopy, Titration, Plaque Assay, Bioassay, Enzyme-linked Immunosorbent Assay, Transfection

Figure Legend Snippet: ( a ) After Myc-MARCH5 WT or Myc-MARCH5 H43W expression vectors were introduced into MARCH5 −/− HEK293T cells, cells were infected with PR8-GFP or transfected with poly(I:C) for 24 h. Promoter activity of IFN-β, ISRE, IFN-α or IRF3. Graphs represent fold-induction relative to the luciferase activity in control cells. Error bars, mean±s.e.m. ( n =3). ( b ) Immunoblot analysis of endogenous or ectopic expression levels of MARCH5 in MARCH5 +/+ or MARCH5 −/− HEK293T cells. See full blots in . ( c , d ) Twenty-four hours after infection, VSV-GFP replication assay by fluorescence microscopy analysis ( c ). Bioassay of IFN-β or IL-6 production (ELISA) ( d ). Error bars, mean±s.e.m. ( n =3). Scale bars, 100 μm. ( e , f ) IRES, MARCH5 WT -Flag or MARCH5 H43W -Flag stably expressing Raw264.7 cells were stimulated by infection with VSV-GFP for 24 h. Twenty-four hours after infection, VSV-GFP replication assay by fluorescence microscopy analysis (left). Quantification of GFP intensity or virus titration by plaque assay (right; e ). Bioassay of IFN-β or IL-6 production (ELISA) in supernatants of cultured Raw264.7 stable cells ( f ). Error bars, mean±s.e.m. ( n =3). All data are representative of at least three independent experiments. Scale bars, 100 μm.

Techniques Used: Expressing, Infection, Transfection, Activity Assay, Luciferase, Control, Western Blot, Fluorescence, Microscopy, Bioassay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Virus, Titration, Plaque Assay, Cell Culture