Fig 1: Npro is recruited to mitochondria after cell stress and protects from apoptosis.(A). MEF cells were transfected with Bax-GFP alone (a–f) or tBid-GFP alone (g–j) or Npro cherry alone (k–n) and either untreated (No NaA, left panel) or treated with sodium arsenate for 4 hours (+NaA, right panel). Cells were incubated with Mitotracker (MT) CM Ros (b,e,i) or Green FM (m) for 30 mins before fixation. (B). Protection from apoptosis induced by sodium arsenate requires IRF3. MEF cells co-expresssing Bax-GFP and either wild type Npro-cherry (a–b) or mutant Npro C112R-cherry (c–d) were treated with sodium arsenate (+NaA). MDBK primary cells, transfected with Bax GFP (e–f) alone or infected with BVDV overnight and then transfected with Bax GFP (g–h), were treated with sodium arsenate (+NaA) for 4 hours. BVDV was detected with bovine hyperimmune serum V182 and detected with Cy3 rabbit anti bovine antibody (red) and DNA was stained with DAPI.
Fig 2: PEI-PDA@C-176 NPs inhibited STING pathway via scavenging dsDNA. (A) Representative immunoblotting of pTBK1, TBK1, pIRF3, IRF3, and STING in BMDMs after indicated treatments (n = 6 independent experiments), using β-Actin as control. ELISA of (B) IFNβ, (C) TNFα and (D) IL-6 revealed that PEI-PDA@C-176 NPs significantly inhibited dsDNA-induced IFNβ, TNFα and IL-6 secretion in human primary fibroblast-like synoviocytes. (E) Intracellular colocalization of dsDNA (red) with PDA and PEI-PDA NPs (green) in RAW264.7 cells. As indicated with white arrows, co-localization of dsDNA and PDA NPs showed up as yellow spots. Scale bars: 5 μm.
Fig 3: Transcription of TLRs signaling components in diestrous and pyometra endometria. A Representative PCR detection of transcripts of TLRs signaling components and of cytokines in diestrous and pyometra endometria. NC negative control. B Relative mRNA expression level (arbitrary units, AU) of IL-1β, IL-6, IL-8, IL-10, and TGFβ evaluated by real-time PCR, in hemolytic E. coli endometria, non-hemolytic E. coli endometria and diestrous endometria. Data are given as mean ± SEM. Columns with different superscripts differ significantly (IL-1β, IL-6, IL-8 a vs b p < 0.01a,b vs c; p < 0.00001; IL-10, TGFβ a vs b p < 0.0001). MyD88 (myeloid differentiation factor 88); TRAM (TRIF related adaptor molecule); TRAF6 (TNFR-associated factor 6); TRIF (TIR domain-containing adaptor inducing interferon (IFN)-β); IRF3 (interferon regulatory factor 3).
Fig 4: Inhibition of apoptosis by Npro correlates with degradation of IRF3.(A). Western blotting of lysates from MEF cell expressing Npro cherry with antibodies against Npro, IRF3 and actin shows loss of IRF3 from cells expressing Npro. Lane 1: Control MEF cells not expressing plasmid. Lane 2: MEF cells stably expressing Npro mcherry. Lane 3 : MEF cells expressing Npro C112R mcherry mutant. Lane 4: MEF cells expressing Npro D136N cherry mutant. Graph shows relative intensities of IRF3 compared to actin for the image scanned with Scion image software http://scion-image.software.informer.com (representative of Western blot repeated three times for each lane). (B). Npro but not Npro C112R inhibits caspase 3/7 activity. MEF cells stably expressing wild type Npro or mutant Npro C112R mcherry were treated with staurosporine, interferon, dsRNA (poly I:C), sodium arsenate (NaA) or hydrogen peroxide (H2O2) for 4 hrs. Fold increase in caspase activity was normalized to negative (untreated cells) for each cell line and analyzed with a two-sided t-test with unequal variance relative to the untransfected cells for each cell line, and significance values are: bars marginally significant (0.05–0.1), * significant (0.01–0.05), ** highly significant (0–0.01) (n = 5 to 8). The table shows significance of drug treatment relative to control treated cells for cells expressing Npro WT or NproC112R and analysis was conducted using similar tests as described above.
Fig 5: IFI44 decreases the kinase activity of IKKβ and IKKε. Human 293T cells were silenced for IFI44, or for FKBP5, and were transfected with plasmids expressing His-IKKε (A) or MYC-IKKβ (B), together with IFI44-HA, and FKBP5-FLAG expression plasmids. At 24 hpt, IKKε (A) and IKKβ (B) complexes were purified with anti-His and anti-MYC antibodies, respectively, and these complexes were assayed in kinase assays using IRF-3 (for the IKKε complexes shown in panel A) and IkBα (for the IKKβ complexes shown in panel B) as substrates. The levels of phosphorylated and unphosphorylated forms of IRF-3 (panel A, bottom blot) and IkBα (panel B, third and fourth blots) were analyzed by Western blotting using specific antibodies. Levels of IKKε were analyzed using an anti-His-specific antibody (A, first blot) and anti-pIKKε (A, second blot), and levels of IKKβ were analyzed using an anti-MYC-specific antibody (B, first blot) and anti-pIKKβ (B, second blot). Western blots were quantified by densitometry using ImageJ software (v1.46). Protein expression levels in cells expressing IKKε (A) and IKKβ (B) alone were assigned a value of 100% for comparisons with the levels of expression in cells expressing the different combinations of IKKε/IFI44/FKBP5 (A) or IKKβ/IFI44/FKBP5 (B) (numbers are indicated below each plot). pIRF-3 and IRF-3 levels (observed in the same bottom blot in panel A) and pIkBα and IkBα (third and bottom blot in panel B) are represented with numbers below each blot. Levels of pIRF-3 and pIkBα normalized to the levels of IKKε and IKKβ are represented in the bottom graphs in panels A and B, respectively. Molecular weight markers are indicated (in kilodaltons) on the right.
Supplier Page from Abcam for Anti-IRF3 antibody