EMBO/SourceData huggingface dataset:
Figure 2Immunisation with cGAMP-VLPs facilitates induction of HIV-1 Gag-specific polyfunctional CD8 T cell responses. Alternatively, cells were analysed by ICS six hours after stimulation with peptide. CD8 T cells were gated as live, CD90.2+, CD8+. CD8 T cells expressing CD107a, IFNγ, TNFα or IL2 were analysed as shown in (E).
Figure 3A, B. cGAMP loading enhances IgG responses specific to VLP proteins, including VSV-G. ELISA plates were coated with lysate from cGAMP-VLPs (A) or recombinant VSV-G protein (B). Antibodies of different isotypes specific for these proteins were measured in sera from immunised mice by ELISA. The optical density at increasing serum dilutions is shown in the first three graphs from the left and the EC50 is on the right.C. Enhanced antibody production following immunisation with cGAMP-VLPs relies on STING signalling. WT or Sting-/- mice were immunised with PBS, cGAMP-VLPs, Empty-VLPs or Empty-VLPs + poly(I:C). IgG2b antibodies recognising VLP proteins were assessed by ELISA. The optical density at increasing serum dilutions is shown in the first two graphs from the left and the EC50 is on the right.
Figure 4Immunisation with cGAMP-VLPs enhances accumulation of Tfh cells in the draining lymph node. T follicular (Tf) cells were identified by flow cytometry as CD4+CD44+CXCR5hiPD1hi cells and were further subdivided into Tfr cells (FoxP3+) and Tfh cells (FoxP3-). the percentages of Tf, Tfh and Tfr cells within CD4+ cells are shown in (B).Immunisation with VLPs induces germinal centre formation. Germinal centre B cells were identified by flow cytometry as B220+IgD-CD95+GL7+ cells. the percentage of germinal centre B cells amongst B220+ cells is shown in (E).
Figure 5C, D. Low doses of cGAMP-HA-VLPs confer protection following IAV challenge. One month after immunisation with the indicated doses of VLPs, animals were infected with 104 TCID50 of IAV PR8 virus. Weight loss was monitored over the following eleven days and is shown as a percentage of starting weight (C, upper graph shows mean and lower graphs show individual mice for each condition). Animals approaching the humane end-point of 20% weight loss were culled and survival to end-point curves are shown in (D).
Figure 6B. cGAMP-HA-VLPs and Empty-HA-VLPs + AddaVax protect against IAV challenge. One month after immunisation, animals were infected with 104 TCID50 of IAV PR8 virus. Animals approaching the humane end-point of 20% weight loss were culled and survival to end-point curves are shown in (B).
Figure 7A. cGAMP-S-VLPs and Empty-S-VLPs incorporate SARS-CoV-2 S. Lysates from VLP producer cells and VLP preparations were analysed by western blot for the presence of the S2 subunit of SARS-CoV-2 S, Gag-GFP and actin using the indicated antibodies.B, C. Immunisation with cGAMP-S-VLPs augments anti-Spike and anti-RBD antibody titres. Mice were immunised via the intra-muscular route with 5x105 IU of cGAMP-S-VLPs, Empty-S-VLPs, or PBS as a control. Three weeks after immunisation, sera were collected, heat-inactivated, and titres of antibodies capable of binding to SARS-CoV-2 S (B) or its RBD (C) were determined by ELISA. The antibody response was expressed as Endpoint titre defined as the reciprocal of the highest serum dilution that gives a positive signal (blank+10SD). The dotted line shows the limit of detection (LOD).D. Immunisation with cGAMP-S-VLPs induces neutralising antibodies. Using serum samples from (B), antibody titres capable of neutralising SARS-CoV-2 were determined by microneutralisation (MN) assay. Calculated IC50 doses from multiple serum dilutions are shown. The dotted line shows the LOD.
Figure 1 A1) Representative WBs showing DRD2 and RGS9-2 downregulation in adult (P60-P90) Tor1a+/- (+/-) striata, characterized by a reduced torsinA protein level with respect to Tor1a+/+ (+/+) littermates. The striatal level of RGS7 is unchanged. The dot plot shows Tor1a+/- data, normalized to Tor1a+/+ controls of the same experiment. TorsinA: Tor1a+/+ N=7, Tor1a+/- N=6, t test ***P=0.0003; DRD2: Tor1a+/+ N=18, Tor1a+/- N=16, t test **P=0.0029; RGS9-2: Tor1a+/+ N=32, Tor1a+/- N=33, t test *P=0.0115). RGS7: Tor1a+/+ N=7, Tor1a+/- N=7, t test P=0.8721). WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type samples of the same experiment. A2,A3,A4) Time-course of changes in torsinA, DRD2 and RGS9-2 striatal levels along postnatal development. Summary plot data are normalized to the Tor1a+/+ P60 sample of each independent experiment. A2) TorsinA level is significantly reduced in mutants throughout the developmental period considered (P7 N=3; P14-P60 N=4; one-way ANOVA P<0.0001 and Bonferroni's Multiple Comparison Test **P<0.01 at P7, P14 and P60). A3,A4) DRD2 and RGS9-2 levels show parallel courses, with a similar increase from P7 to P21 in Tor1a+/+ as well as Tor1a+/- striatal lysates (P7: DRD2 N=3, RGS9-2 N=4; P14-P21 N=4; one-way ANOVA with Bonferroni's Multiple Comparison Test P>0.05). At P60, a simultaneous reduction of DRD2 and RGS9-2 levels is observed in Tor1a+/- striatal lysates (N=4; Tor1a+/- one sample t test DRD2: *P=0.0250, RGS9-2: *P= 0.0175). WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type P60 sample of the same experiment B) Representative WB images and the dot plot show downregulation of torsinA, DRD2 and RGS9-2 striatal levels also in adult P60-P90 Tor1a∆gag/+ (∆gag/+) mice. TorsinA: Tor1a+/+ N=10, Tor1a∆gag/+ N=12, t test ***P= 0.0006; DRD2: Tor1a+/+ N=7, Tor1a∆gag/+ N=9, t test *P= 0.0183; RGS9-2: Tor1a+/+ N=10, Tor1a∆gag/+ N=9, t test *P= 0.0192. WB quantification data, expressed as the ratio of protein vs. loading control intensity level, are normalized to the wild-type samples of the same experiment. Mean ± SEM is represented in the graphsC) Left. Representative image of coronal striatal slices of fresh frozen brains of wild-type and Tor1a+/- mice probed with the DRD2 radioligand 3H-spiperone. Scale bar 1.5 mm. (Right) Graph showing DRD2 binding density in Tor1a+/- striatal sections, obtained from the densitometric quantification analysis, expressed as percentage variation compared to control animals
Figure 2A1) Representative WB of RGS9-2, R7BP, and Gβ5 proteins on four Tor1a+/+ and four Tor1a+/- striatal lysates. The bar histogram on the right shows the quantification of the three proteins in each sample. A2) Dot plot showing that Gβ5 striatal levels are unchanged in Tor1a+/- mice (Tor1a+/+ N=11, Tor1a+/- N=10, Mann Whitney test P=0.2453). A3) Conversely, the level of the R7 binding protein R7BP is increased in Tor1a+/- striatum (Tor1a+/+ N=12, Tor1a+/- N=12, t test **P=0.0014). Values are represented as ratio of protein vs. loading control intensity level, normalized to the mean of Tor1a+/+ control values of the same experimentB1) Representative WB of lysates of Tor1a+/+ and Tor1a+/- dorsal striatum slices incubated for 5 hours in the presence (w/ leu) or absence (w/o leu; contralateral striatum) of the protease inhibitor leupeptin. Samples showing enhanced degradation of DRD2 and/or RGS9-2 proteins show additional bands at lower molecular weight. B2) Summary plot reporting mean ± SEM of RGS9-2 and DRD2 protein level values, expressed as the ratio of protein vs. loading control intensity level, normalized to the value of the Tor1a+/+ w/leu sample measured in the same experiment. DRD2: Tor1a+/+ N=4, t test P=0.4376; Tor1a+/- N=4, t test *P=0.0427; RGS9-2: Tor1a+/+ N=5, t test *P=0.0464; Tor1a+/- N=5, t test *P= 0.0190C1, C2, C3, C4) Dot plots showing DRD2, RGS9-2, R7BP and Gβ5 protein levels measured in striatal detergent-resistant-membrane (DRM) preparations from Tor1a+/- (+/-) and WT (+/+) mice (DRD2: Tor1a+/+ N=16, Tor1a+/- N=13, t test P=0.0129; RGS9-2: Tor1a+/+ N=7, Tor1a+/- N=6, t test *P=0.0396; R7BP: Tor1a+/+ N=6, Tor1a+/- N=6, t test ***P=0.0002; Gβ5: Tor1a+/+ N=9, Tor1a+/- N=9, t test P=0.8461)
Figure 3WB analysis of markers of endocytic trafficking and degradative pathway in the dorsal striatum of Tor1a+/- mice. A) The level of β-arrestin 2 (β-Arr2) is not altered (Tor1a+/+ N=11, Tor1a+/- N=12, t test P=0.6796), whereas the level of spinophilin (B) is decreased (Tor1a+/+ N=5, Tor1a+/- N=8, t test *P=0.0151)C) A Sh-RGS9-2-GFP viral preparation injected into the right striatum (+) of C57Bl/6 mice effectively reduces both RGS9-2 and DRD2 protein levels, with respect to the contralateral striatum (-).The summary plot reports mean ± SEM values (RGS9-2: N=4, paired t test *P=0.0100; DRD2: N=4, paired t test *P=0.0432)D) The endosomal marker Rab4 is upregulated in the Tor1a+/- striatum (Tor1a+/+ N=13, Tor1a+/- N=14, t test **P=0.0087)E) Basal levels of markers of the degradative pathway, p62 and LC3-II, show a trend towards upregulation in Tor1a+/- striatal lysates (p62: Tor1a+/+ N=11, Tor1a+/- N=11, t test P=0.2292; LC3-II: Tor1a+/- N=9, Tor1a+/- N=11, t test P=0.1574)F) Treatment with bafilomycin A1 (BafA1) induces a significantly more pronounced increase of LC3-II in Tor1a+/- than in wild-type dorsal striatum slices. BafA1-induced increase in LC3-II level measured in Tor1a+/- slices was normalized to the increase observed in the Tor1a+/+ samples of the same experiment (N=4, one sample t test **P=0.0012)G) LAMP-2, a lysosomal marker, shows a trend for upregulation (Tor1a+/+ N=8; Tor1a+/- N=6, t test P= 0.2232)
Figure 5A) Top. Low magnification merged confocal images of coronal striatal sections showing ChAT-labelled (cyano) ChIs in the area of mCherry fluorescence (red) indicating viral infection. Bar = 100 µm. Bottom. Higher magnification images of the ChIs identified by the white box in the upper images show viral-induced expression of mCherry and GFP signal. Bar = 20 µm. B) The mCherry/ChAT overlap is increased in ChIs from Tor1a+/- mice (Tor1a+/+ n=24 cells, Tor1a+/- n=22 cells, N=8 mice/genotype, t test ***P=0.0005), but the mCherry/GFP fluorescence ratio indicated that the LC3 flux was unchanged (Tor1a+/+ n=19 cells, Tor1a+/- n=21 cells, N=8 mice/genotype, Mann Whitney test P=0.2334)C) Merged and split channels confocal images (bar = 10 µm) of representative Tor1a+/+ and Tor1a+/- ChIs showing red dots and yellow puncta (indicated by white circles), quantified in (D): red dots: Tor1a+/+ n=13 cells, Tor1a+/- n=14 cells, t test ***P=0.0003; yellow dots: Tor1a+/+ n=13 cells, Tor1a+/- n=14 cells, t test **P=0.0048, N=5 mice/genotype). Measurement of the mCherry/yellow dots ratio indicated that the autophagic flux was not altered (Tor1a+/+ n=13 cells; Tor1a+/- n=14 cells, Mann Whitney test P=0.8842). Data in B,D) are represented as mean ± SEM
Figure 7A) Middle. Graphic reconstruction of an anteroposterior sequence of coronal sections of mouse striatum, reporting the coordinates relative to Bregma. HSV-LacZ (left, grey) and HSV-RGS9-2 (right, red) were injected into the left and right striatum, respectively, and mice sacrificed after 3-5 days. Left. Serial confocal images showing the extent of viral infection, as indicated by the robust dark β-gal staining. Right. The RGS9-2 immunolabelling (red) is particularly intense near the injection site, indicating enhanced protein expression with respect to the endogenous level. Bar = 200 µmB) A representative WB shows correlated viral-mediated increases in RGS9-2 and DRD2 protein levels in Tor1a+/- dorsal striata (lanes 4 and 5) . while in a sample where RGS9-2 level was not increased by viral infection (lane 6) DRD2 level remained low as in the sham-injected striatum (lane 7). C,D) The summary plots report mean ± SEM values of the ratio of protein vs. loading control intensity level measured from the injected (HSV/LV) or sham contralateral Tor1a+/- (+/-) striata, normalized to the Tor1a+/+ (+/+) samples of the same experiment. C) RGS9-2: Tor1a+/+ (N=10); Tor1a+/- injected (N=7) vs. contralateral (N=7): paired t test **P=0.0011; D) DRD2: Tor1a+/+ (N=9); Tor1a+/- injected (N=6) vs. contralateral (N=6): paired t test *P=0.0380
Figure 8A) As depicted in the cartoon and shown by representative confocal images, patch-clamp recordings were performed in the dorsal striatum, in the area of HSV-LacZ (blue, left) and HSV-RGS9-2 (red, right) viral infection, the latter showing an increased RGS9-2 immunolabelling with respect to the sham contralateral striatum of the same mouse. Representative confocal images at higher magnification show colocalization of RGS9-2 immunolabeling with Lucifer Yellow (LY) fluorescence in two ChIs loaded intracellularly with LY during whole-cell recordings in the infected areaB) Representative patch-clamp recordings of ChIs from hMT (top, perforated configuration) and Tor1aΔgag/+ (bottom, cell-attached configuration) mice, showing that viral-mediated delivery of RGS9-2 rescues the physiological response to the DRD2 agonist quinpirole (10 µM, 2 min). The dot plot reports the firing frequency changes induced by quinpirole in hMT striata (N=6, HSV-RGS9-2 n=9, HSV-LacZ n=14, t test P<0.0001)C) Representative traces show a typical response of Tor1a+/+ ChIs to quinpirole, characterized by a reduction in the frequency of the ongoing firing activity of the cell, as opposed to an aberrant increase in firing frequency in Tor1a+/- ChIs, quantified in the plot (right, Tor1a+/+ N=6, n=14; Tor1a+/- N=10, n=27; Tor1a+/+ vs. Tor1a+/- in quinpirole t test ***P=0.0005; Tor1a+/- basal vs. quinpirole paired t test ***P=0.0009)D) LV-mediated delivery of RGS9-2 rescues the physiological response to quinpirole in Tor1a+/- slices. Dot plot reporting firing frequency changes induced by quinpirole in Tor1a+/+ (N=3) and Tor1a+/- (N=6) striatal ChIs infected with LV/HSV-RGS9-2 or control LV-GFP and HSV-LacZ ChIs (Tor1a+/+ RGS9-2 n=7; Tor1a+/- RGS9-2 n=9; Tor1a+/- LacZ/GFP n=8; Tor1a+/-: LacZ/GFP vs. RGS9-2 t test *P=0.0136)
Figure 1C. HEK293 cells were incubated with 5 µg of purified EVCx43- or EVCx43+ for 24 h, in the presence or absence of Bafilomycin A1 (Baf) in the last 6 h. The levels of LC3 were analyzed by WB and are depicted on graph (LC3-II; n=4 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.E. HEK293 cells were incubated with EVCx43- or EVCx43+ for 24 h before WB analysis of ATP2A2/SERCA2, a predicted target of miRNAs enriched in Cx43-containing EV. Graph depicts quantification of ATP2A2/SERCA2 levels (n=4 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.
Figure 3A. Cx43 was immunoprecipitated (IP) from HEK293Cx43+ cells, after which interaction with hnRNPA2B1 and hnRNPQ was analyzed by WB.B. Co-localization between Cx43 (green) and hnRNPA2B1 or hnRNPQ (red) in C33a cells was analyzed by confocal microscopy. Scale bars 10 μm. Arrowheads in the inset images highlight co-localization between Cx43 and hnRNPs.
Figure 5A. Cell free EV biogenesis was assessed by relative protection of Cx43-luciferase. Reactions were incubated at 4ºC or 30ºC in the presence of membranes derived from HEK293Cx43- transiently transfected with Cx43-luciferase and cytosol from HEK293Cx43- cells, except in the negative control. 1% TX100 was used to disrupt membranes, where indicated (n=3 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.B. Crude membranes and cytosol from broken HEK293Cx43+ or HEK293Cx43- cells were mixed in an ATP regenerating system, supplemented with synthetic miR-133b or miR-509-3p, as indicated. Graph depicts the indicated miRNAs levels, assessed by RT-qPCR and represented as percent protected after RNAse I digestion of unincorporated miRNAs (n=7-8 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.C. Co-immunoprecipitation of endogenous miR-133b and miR-509-3p following UV crosslinking immunoprecipitation (CLIP) of Cx43 (n=3-5 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.D. WB analysis for Cx43 in samples derived by miRNAs pulldowns performed with whole lysates of HEK293Cx43+ cells and biotinylated miR-133b and miR-509-3p (n=5 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.EMSA was performed using either Cy3-labeled miR-133b (E , and Cx43 produced by in vitro translation inserted into lipid nanodiscs, For competition EMSA, an excess of unlabeled miR-133b was used (competitor). Empty nanodiscs were used to control unspecific binding.EMSA was performed using either Cy3-labeled miR-133b F) and Cx43 produced by in vitro translation inserted into lipid nanodiscs, in the presence or absence of recombinant GST-hnRNPA2B1 or GST-hnRNPQ, as indicated.EMSA was performed using either Cy3-labeled miR-509-3p (G and Cx43 produced by in vitro translation inserted into lipid nanodiscs, For competition EMSA, an excess of unlabeled miR-133b or miR-509-3p was used (competitor). Empty nanodiscs were used to control unspecific binding.EMSA was performed using either Cy3-labeled miR-509-3p H) and Cx43 produced by in vitro translation inserted into lipid nanodiscs, in the presence or absence of recombinant GST-hnRNPA2B1 or GST-hnRNPQ, as indicated.I. EMSA was performed using either Cy3-labeled wild type or mutant miR-199a-3p, and Cx43-enriched membranes prepared from C33a parental cells (Cx43+). Cx43- membranes were used to control unspecific binding.
Figure 6Representative maximum projection images of microscopy-based miRNA-protein interaction using beads coated with GST-tagged NT/CT-Cx43, hnRNPA2B1 or hnRNPQ, as indicated, following the addition of Cy3-labelled miR-133b or miR-509-3p. Graph depicts quantification of total fluorescence/bead, as a measure of protein-miRNAs binding. Scale bars 50 μm (n=2 biological replicates; average of 23 beads analyzed/condition). Data information: p-values were derived by Kruskal-Wallis (Dunn's post-hoc) test. Bars and error bars indicate mean ± SD in all graphs.
Figure 7A. Equal amounts of EVCx43+ and EVCx43- labelled with SYTO® RNASelect™ green fluorescent stain were incubated with HEK293Cx43+ cells for 30 min, at 37ºC. Scale bars 10 μm (n=5 biological replicates). p-values were derived by Mann-Whitney test.
Figure 1MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intravenously infected with VSV-GFP (2 × 108 PFU/mouse), and serum samples were collected at 12 hpi (hours post-infection). (A) Virus titer was analyzed in a plaque assay.MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intravenously infected with VSV-GFP (2 × 108 PFU/mouse), and serum samples were collected at 12 hpi (hours post-infection). (B) Secretion of IFN-β, IL-6, IL-12, and TNFα was measured in specific ELISAs.C MARCH2+/+ (n = 6) or MARCH2-/- (n = 6) mice were intravenously injected with poly(I:C) (200 μg/mouse). Serum samples were collected at 2 hpi and secretion of IFN-β, IL-6, IL-12, and TNFα was measured in specific ELISAs.MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. (D) The percentage of surviving mice (D, log-rank test, **P < 0.01)MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. body weight changes for each group (E) are shown.MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. (F, G) At 3 dpc, the bacterial load in the spleen (F) and liver (G) was examined.MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. (H-J) Amount of IL-6, IL-12, CCL5, and CXCL10 in serum (H), spleen (I), and liver (J) of mice in each group at 24 hpc.
Figure 2MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were challenged intraperitoneally with LPS (24 mg/kg). Percentage of surviving mice (A, log-rank test, **P < 0.01)MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were challenged intraperitoneally with LPS (24 mg/kg). body weight changes (B) in each groups.C MARCH2+/+ (n = 4) or MARCH2-/- (n = 4) mice were challenged intraperitoneally with LPS (24 mg/kg). Representing slides of H&E staining of spleen sections from each group. Scale bar, 6mm.D MARCH2+/+ (n = 6) or MARCH2-/- (n = 6) mice were challenged intraperitoneally with LPS (24 mg/kg). Levels of IL-6, TNF-α, CXCL-10, CCL-5 in serum from mice in each groups were measured at12 hpc by ELISA.E, F MARCH2+/+ (n = 5) or MARCH2-/- (n = 5) mice were challenged intraperitoneally with LPS (24 mg/kg). cDNA was prepared from total RNA extracted from spleen and liver of mice. Expression of mRNA encoding IL-6, TNF-α, CXCL10, IL-1β in spleen (E) and liver (F) from mice in each group was examined at 6 hpc by qPCR.
Figure 3A-C BMDMs isolated from MARCH2+/+ or MARCH2-/- mice were infected with PR8-GFP (A, MOI=3), VSV-GFP (B, MOI = 5), or HSV-GFP (C, MOI = 3), and virus titer was measured in a plaque assay.D, E BMDMs isolated from MARCH2+/+ or MARCH2-/- mice were infected with viruses or treated with poly(I:C) (80 µg/ml) or poly (dA:dT) (1 µg/ml). The concentration of secreted IFN-β (D) and IL-6 (E) in the supernatants was measured in an ELISA.PBMCs isolated from whole peripheral blood from MARCH2+/+ or MARCH2-/- mice were infected with VSV-GFP (MOI =1). (F) The virus titer was measured in a plaque assay.PBMCs isolated from whole peripheral blood from MARCH2+/+ or MARCH2-/- mice were infected with VSV-GFP (MOI =1). (G) Concentration of IFN-β and IL-6 in the supernatant was measured in an ELISA.PMs isolated from MARCH2+/+ or MARCH2-/- mice infected with CVB-GFP (MOI = 3). (H) Virus titer was measured in a plaque assay.PMs isolated from MARCH2+/+ or MARCH2-/- mice infected with CVB-GFP (MOI = 3). (I) Concentration of IFN-β and IL-6 in the supernatant was measured in an ELISA.J, K BMDMs (J) or PMs (K) isolated from MARCH2+/+ or MARCH2-/- mice were infected with Salmonella typhimurium or L. monocytogenes, or treated with LPS or zymosan. The concentration of IL-6 and IL-12 in supernatant was analyzed in an ELISA.L, M RAW 264.7 cells transfected with control siRNA (si-control) or MARCH2-specific siRNA (si-MARCH2) were infected with PR8-GFP (MOI = 1), VSV-GFP (MOI = 0.5), poly (I:C) (20 µg/ml) or HSV-GFP (MOI = 1) poly (dA:dT) (1 µg/ml) and IFN-β (L), and IL-6 (M) levels in the supernatant were measured in an ELISA.N RAW 264.7 cells transfected with control siRNA (si-control) or MARCH2-specific siRNA (si-MARCH2) were infected with S. typhimurium, or L. monocytogenes, or treated with LPS (100 ng/ml) or zymosan (100 µg/ml) and IL-6 secretion into the cell supernatant was measured in an ELISA