Confirmation of MATα2 and BHMT interaction.

<p>Cos7 cells were transfected with pFLAG-BHMT, pHA-MATα2 or cotransfected with both plasmids at a 1:1 (w/w) ratio. Lysates were obtained 48 hours posttransfection and immediately processed by anti-FLAG immunoprecipitation. Input (25 μl) and immunoprecipitate samples (40 μl) were analyzed by western blotting using specific antibodies and mouse TrueBlot. (A) Representative anti-HA results from a single immunoprecipitation experiment (N = 8). (B) Representative anti-FLAG immunoblot from a single immunoprecipitation experiment (N = 8). The size of the protein standards is indicated on the left side of each image. (C) Anti-HA signals were quantified using ImageJ and the immunoprecipitate/input ratio (mean ± SEM) for all the experiments calculated (N = 8) to correct for differences in expression between control bearing only pHA-MATα2 and cotransfectants. Statistical analysis of the data was performed by Student’s t-test using GraphPad Prism; *p≤0.05.</p

IFN and VACV infection increases proinflammatory cytokine levels in <i>ISG15</i>^-/- BMDM and increases arginase-1 activity.

<p><b>(A)</b><i>ISG15</i>^+/+ or <i>ISG15</i>^-/- BMDM were infected with VACV (1 PFU/cell). Cellular lysates collected at 2 and 6 hpi, or from mock-infected cultures, were analyzed by 12% SDS-PAGE, transferred to nitrocellulose membranes, and the expression of iNOS, Arg-1, ISG15 or β-actin (protein loading control) was examined by western blotting using specific antibodies. Uninfected M1 or M2 polarized <i>ISG15</i>^-/- BMDM were used as iNOS or Arg-1 controls. <b>(B)</b> Under the same conditions as above, the production of urea was measured as a marker of Arg-1 activity. The reaction was performed following the indications of the manufacturer. Results represent the mean ± the standard deviation of five biological replicates. <b>(C)</b> The expression level of TNF-α, IFN-β, IL-6, IL-1β and IL-12 genes was measured by quantitative RT-PCR. Triplicate samples were measured in three independent experiments; data shown is representative of one experiment. <b>(D)</b> IL-6 levels in the medium of <i>ISG15</i>^+/+ and <i>ISG15</i>^-/- BMDM were quantified by ELISA. Aliquots (100 μl) of supernatant from <i>ISG15</i>^+/+ or <i>ISG15</i>^-/- BMDM uninfected or at 2, 6, hpi were used for ELISA according to the manufacturer's instructions. Triplicate samples were measured in two independent experiments.</p

Interactions reported for BHMT in human, rat or mouse.

<p>Interactions reported for BHMT in human, rat or mouse.</p

Comparative proteomics analysis of <i>ISG15</i>^-/- <i>versus ISG15</i>^+/+ IFN-treated BMDM.

<p>Ingenuity pathway analysis showing selected canonical pathways differently modulated in <i>ISG15</i>^-/- <i>versus ISG15</i>^+/+ positives values IFN-treated BMDM (p < 0.05). Percentage of proteins down- or up-regulated in selected canonical pathways differently modulated in <i>ISG15</i>^-/- <i>versus ISG15</i>^+/+ BMDM after pretreatment with IFN (500 units/ml, 16 hours) (p < 0.05).</p

Elution profiles from control, intein and intein-BHMT loaded chitin columns.

<p>The figure shows representative A₂₈₀ elution profiles from control (beads only; 4 ml), intein (4 ml) and intein-BHMT (1 ml) loaded chitin columns, as well as representative Coomassie Blue stained SDS-PAGE gels of liver cytosol, flowthroughs loaded on these columns and samples of the eluted peaks. (A) Control columns were loaded with liver cytosol and eluted with a NaCl gradient. (B) The flowthrough from the control column was loaded onto the intein column and elution performed with the same salt gradient. (C) The flowthrough of the intein column was loaded onto the intein-BHMT column and elution carried out with a NaCl gradient. (D) SDS-PAGE of the protein fractions loaded onto the columns: liver cytosol (2 μl); flowthrough of the chitin column (2 μl); flowthrough of the intein column (2 μl); and flowthrough of the intein BHMT column (2 μl). (E) Eluted proteins using salt gradients and 2-mercaptoethanol (2-ME) excision were collected and analyzed by SDS-PAGE: chitin peak (40 μl); intein peak (40 μl); and intein-BHMT peak (40 μl). Molecular weight standards are shown on the right side of each gel.</p

Corroboration of the BHMT/actin B interaction.

<p>Cos7 cells were transfected with pFLAG-BHMT, pHA-actin B or cotransfected with both plasmids at a 1:1 (w/w) ratio. Lysates were obtained 48 hours posttransfection and immediately processed by anti-FLAG immunoprecipitation. Input (25 μl) and immunoprecipitate samples (40 μl) were analyzed by western blotting using specific antibodies and mouse TrueBlot. (A) Representative anti-HA immunoblot from the immunoprecipitations experiments carried out (N = 7). (B) Representative anti-FLAG immunoprecipitations from the experiments performed (N = 7). The size of the protein standards is indicated on the left side of each image. (C) Anti-HA signals were quantified using ImageJ and the immunoprecipitate/input ratio (mean ± SEM) from all the experiments performed (N = 7) calculated to correct for differences in expression between control bearing only pHA-actin B and cotransfectants. Statistical analysis of the data was performed by Student’s t-test using GraphPad Prism; *p≤0.05.</p

Hepatic BHMT interaction targets identified by yeast two-hybrid.

<p>Hepatic BHMT interaction targets identified by yeast two-hybrid.</p

STRING classification of BHMT interaction targets according to molecular function (GO).

<p>STRING classification of BHMT interaction targets according to molecular function (GO).</p

Coimmunoprecipitation of Prkaca and BHMT.

<p>Cos7 cells were transfected with pFLAG-BHMT, pHA-Prkaca or cotransfected with both plasmids at a 1:1 (w/w) ratio. Lysates were obtained 48 hours posttransfection and immediately processed by anti-FLAG immunoprecipitation. Input (25 μl) and immunoprecipitate samples (40 μl) were analyzed by western blotting mouse primary antibodies and mouse TrueBlot. (A) Representative anti-HA immunoblot of the immunoprecipitation experiments performed (N = 3). (B) Representative images of anti-FLAG immunoblots of the experiments carried out (N = 3). The size of the protein standards is indicated at the left side of each image. (C) Anti-HA signals were quantified from all experiments (N = 3) using ImageJ and the immunoprecipitate/input ratio (mean ± SEM) calculated to correct for differences in expression between control bearing only pHA-Prkaca and cotransfectants. Statistical analysis of the data was performed by Student’s t-test using GraphPad Prism; *p≤0.05.</p

Defective pro-inflammatory cytokine response to <i>Lm</i> in <i>Hdac6</i>^<i>-/-</i> BMDCs.

<p>A) PCR analysis of type-I interferons (PanIFN-α and IFN-β), interferon downstream proteins (Mx1, IFIT3 and ISG15), pro-inflammatory cytokines (TNF-α, IL-1β and IL-12p40) chemokine receptor (CXCR1) and chemokines (CXCL5 and CXCL10) of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs non-infected (NI) and infected with <i>Lm</i> at 6 hpi (arbitrary units). ***p≤0.001, ** p≤0.01, * p≤0.05; n = 5–6. B) ELISA analysis of the pro-inflammatory cytokines TNFα, IL1β, IL6 and IL12p70 (pg/ml) and IFN-β in supernatants of <i>Hdac6</i>^<i>+/+</i> and <i>Hdac6</i>^<i>-/-</i> BMDCs at 6, 12 and 24 hpi with <i>Lm</i>. ***p≤0.001, ** p≤0.01, * p≤0.05 ns>0.05 non-significant; n = 5–6.</p