Journal: Oncogene

Article Title: K63-ubiquitylation of VHL by SOCS1 mediates DNA double-strand break repair.

doi: 10.1038/onc.2013.22

Figure Lengend Snippet: Figure 1. SOCS1 binds VHL in the nucleus. (a) U2OS cells were transfected with the indicated plasmids and immunostained with polyclonal anti-HA (red) and monoclonal anti-FLAG (green) antibodies. Blue, DAPI (4,6-diamidino-2-phenylindole) staining. (b) HEK293A cells transfected with the indicated plasmids were fractionated into cytoplasmic and nuclear pools and immunoblotted with the indicated antibodies or (c) fractionated, immunoprecipitated (IP) with monoclonal anti-HA, and immunoblotted (IB) with polyclonal anti-HA and anti-FLAG (top). Unmodified, IP HA-VHL is shown in the lower anti-HA panel with a short exposure (SE) and higher-molecular-weight-modified VHL (HA-VHL- Mod(n)) is shown in the upper anti-HA panel with a long exposure (LE). Equal amounts of cytoplasmic or nuclear fractions (Inputs) were also IB with the indicated antibodies (bottom).

Article Snippet: Antibodies The following antibodies were used: monoclonal anti-FLAG(M2) (SigmaAldrich, Oakville, ON, Canada ), polyclonal anti-HA (Cell Signaling, Danvers, MA, USA), monoclonal anti-HA(12CA5) (Roche Applied Science, Laval, QC, Canada), anti-hnRNP C1/C2 (AbCam, Cambridge, MA, USA), anti-vinculin (Sigma-Aldrich), polyclonal anti-FLAG (Novus Biologicals, Oakville, ON, Canada), anti-a-tubulin (Sigma-Aldrich), anti-VHL (BD Biosciences, Mississauga, ON, Canada), anti-ubiquitin (Dako, Burlington, ON, Canada), antiT7 (Novagen, Madison, WI, USA), anti-gH2AX (immunoblotting; Cell Signaling), anti-gH2AX (immunofluorescence; Millipore, Billerica, MA, USA), anti-SOCS1 (Invitrogen, Burlington, ON, Canada), anti-Ubc13 (Cell Signaling), anti-pChk1 (Cell Signaling), anti-pATM(Ser1981) (Cell Signaling), anti-ATM (Cell Signaling), anti-Mre11 (Genetex, Irvine, CA, USA), anti-HIF2a (Novus Biologicals) and anti-b-actin (AbCam).

Techniques: Transfection, Staining, Immunoprecipitation, Molecular Weight

Journal: Oncogene

Article Title: K63-ubiquitylation of VHL by SOCS1 mediates DNA double-strand break repair.

doi: 10.1038/onc.2013.22

Figure Lengend Snippet: Figure 2. SOCS1 promotes covalent modification of VHL on lysine residues. (a–c) Lysates from HEK293A cells transfected with the indicated plasmids were immunoprecipitated (IP) with the monoclonal antibodies indicated and immunoblotted (IB) with the indicated polyclonal antibodies (top). Equal amounts of cell lysates were immunoblotted with anti-HA or anti-FLAG and anti-a-tubulin (bottom). *Nonspecific protein band. LE, long exposure; SE, short exposure; WCE, whole-cell extract.

Article Snippet: Antibodies The following antibodies were used: monoclonal anti-FLAG(M2) (SigmaAldrich, Oakville, ON, Canada ), polyclonal anti-HA (Cell Signaling, Danvers, MA, USA), monoclonal anti-HA(12CA5) (Roche Applied Science, Laval, QC, Canada), anti-hnRNP C1/C2 (AbCam, Cambridge, MA, USA), anti-vinculin (Sigma-Aldrich), polyclonal anti-FLAG (Novus Biologicals, Oakville, ON, Canada), anti-a-tubulin (Sigma-Aldrich), anti-VHL (BD Biosciences, Mississauga, ON, Canada), anti-ubiquitin (Dako, Burlington, ON, Canada), antiT7 (Novagen, Madison, WI, USA), anti-gH2AX (immunoblotting; Cell Signaling), anti-gH2AX (immunofluorescence; Millipore, Billerica, MA, USA), anti-SOCS1 (Invitrogen, Burlington, ON, Canada), anti-Ubc13 (Cell Signaling), anti-pChk1 (Cell Signaling), anti-pATM(Ser1981) (Cell Signaling), anti-ATM (Cell Signaling), anti-Mre11 (Genetex, Irvine, CA, USA), anti-HIF2a (Novus Biologicals) and anti-b-actin (AbCam).

Techniques: Transfection, Immunoprecipitation, Bioprocessing

Journal: Oncogene

Article Title: K63-ubiquitylation of VHL by SOCS1 mediates DNA double-strand break repair.

doi: 10.1038/onc.2013.22

Figure Lengend Snippet: Figure 3. SOCS1 induces K63-ubiquitylation of VHL. (a) HEK293A cells transfected with the indicated plasmids were lysed in the absence of MG132 and immunoblotted (IB) with the indicated antibodies (left) or immunoprecipitated (IP) with monoclonal anti-HA and IB with polyclonal anti-HA (top panel) or anti-ubiquitin (bottom panel). *Nonspecific protein band. (b) HEK293A cells transfected with the indicated plasmids were treated with 5 mg/ml MG132 for 4 h before lysis. Cell lysates were IP with anti-T7 and IB with the indicated antibodies (top). Equal amounts of cell lysates were also IB with the indicated antibodies (bottom). (c) Lysates from HEK293A cells transfected with the indicated plasmids were IP with anti-T7 and IB with the indicated antibodies (top). Equal amounts of cell lysates were also IB with anti-FLAG and anti-a-tubulin (bottom). LE, long exposure; SE, short exposure; WCE, whole-cell extract.

Article Snippet: Antibodies The following antibodies were used: monoclonal anti-FLAG(M2) (SigmaAldrich, Oakville, ON, Canada ), polyclonal anti-HA (Cell Signaling, Danvers, MA, USA), monoclonal anti-HA(12CA5) (Roche Applied Science, Laval, QC, Canada), anti-hnRNP C1/C2 (AbCam, Cambridge, MA, USA), anti-vinculin (Sigma-Aldrich), polyclonal anti-FLAG (Novus Biologicals, Oakville, ON, Canada), anti-a-tubulin (Sigma-Aldrich), anti-VHL (BD Biosciences, Mississauga, ON, Canada), anti-ubiquitin (Dako, Burlington, ON, Canada), antiT7 (Novagen, Madison, WI, USA), anti-gH2AX (immunoblotting; Cell Signaling), anti-gH2AX (immunofluorescence; Millipore, Billerica, MA, USA), anti-SOCS1 (Invitrogen, Burlington, ON, Canada), anti-Ubc13 (Cell Signaling), anti-pChk1 (Cell Signaling), anti-pATM(Ser1981) (Cell Signaling), anti-ATM (Cell Signaling), anti-Mre11 (Genetex, Irvine, CA, USA), anti-HIF2a (Novus Biologicals) and anti-b-actin (AbCam).

Techniques: Transfection, Immunoprecipitation, Ubiquitin Proteomics, Lysis

Journal: Oncogene

Article Title: K63-ubiquitylation of VHL by SOCS1 mediates DNA double-strand break repair.

doi: 10.1038/onc.2013.22

Figure Lengend Snippet: Figure 4. SOCS1 mediates K63-uiquitylation of VHL in response to DSBs. (a) HEK293A cells were transfected with the indicated plasmids. 24 h post transfection, cells were treated with ( þ ) or without ( ) 300 ng/ml doxorubicin for 16 h. Lysates were immunoprecipitated (IP) with monoclonal anti-HA, and immunoblotted with polyclonal anti-HA (top). Equal amounts of whole-cell extracts (WCEs) were also immunoblotted (IB) with the indicated antibodies (bottom). (b) 786-VHL(WT)-shScr and -shSOCS1 cells were treated with ( þ ) or without ( ) 300 ng/ml doxorubicin for 16 h. Lysates were IP with monoclonal anti-HA and IB with polyclonal anti-HA (top). RNA was also extracted and mRNA levels of SOCS1 were determined by quantitative real-time PCR and normalized to b-Actin. The relative level of SOCS1 transcript levels in 786-VHL-shScr cells was set to 1.0 (bottom). (c) 786-VHL(WT) cells were transfected with siScr or siUbc13. Twenty-four hours post transfection, cells were treated with ( þ ) 300 ng/ml doxorubicin for 16 h. Lysates were IP with monoclonal anti-HA and IB with polyclonal anti- HA (top). Equal amounts of cell lysates were IB with anti-Ubc13 and anti-a-tubulin (bottom). LE, long exposure; SE, short exposure.

Article Snippet: Antibodies The following antibodies were used: monoclonal anti-FLAG(M2) (SigmaAldrich, Oakville, ON, Canada ), polyclonal anti-HA (Cell Signaling, Danvers, MA, USA), monoclonal anti-HA(12CA5) (Roche Applied Science, Laval, QC, Canada), anti-hnRNP C1/C2 (AbCam, Cambridge, MA, USA), anti-vinculin (Sigma-Aldrich), polyclonal anti-FLAG (Novus Biologicals, Oakville, ON, Canada), anti-a-tubulin (Sigma-Aldrich), anti-VHL (BD Biosciences, Mississauga, ON, Canada), anti-ubiquitin (Dako, Burlington, ON, Canada), antiT7 (Novagen, Madison, WI, USA), anti-gH2AX (immunoblotting; Cell Signaling), anti-gH2AX (immunofluorescence; Millipore, Billerica, MA, USA), anti-SOCS1 (Invitrogen, Burlington, ON, Canada), anti-Ubc13 (Cell Signaling), anti-pChk1 (Cell Signaling), anti-pATM(Ser1981) (Cell Signaling), anti-ATM (Cell Signaling), anti-Mre11 (Genetex, Irvine, CA, USA), anti-HIF2a (Novus Biologicals) and anti-b-actin (AbCam).

Techniques: Transfection, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Fish & shellfish immunology

Article Title: Involvement of N-acetylneuraminate cytidylyltransferase in Edwardsiella piscicida pathogenicity.

doi: 10.1016/j.fsi.2022.04.033

Figure Lengend Snippet: Fig. 5. Enhancement of sialylation in epCSS. (A) Sialic acid contents in E. piscicida were obtained using CM-soluble and -insoluble fractions. These fractions were hydrolyzed with H2SO4 and the released sialic acids were analyzed by HPLC. n = 6. (B–D) Lysates of E. piscicida were employed for lectin blotting using MAM lectin (B), SSA lectin (C), and MAL II lectin (D). (E, F) Western blotting of E. piscicida lysate was conducted using the anti-PSA antibody (E) and anti-flagellin antibody (F).

Article Snippet: Flagellin and polysialic acid (PSA) were detected by incubation with anti-flagellin antibody (1/1000 diluted, Rockland Immunochemicals, Inc, PA) or anti-PSA (1/1000 diluted, clone 735, Abnova, Taiwan), followed by reaction with secondary HRP anti-rabbit IgG antibody (1:10000).

Techniques: Western Blot

Journal: Vaccine

Article Title: Self-assembling protein nanoparticles with built-in flagellin domains increases protective efficacy of a Plasmodium falciparum based vaccine.

doi: 10.1016/j.vaccine.2017.12.001

Figure Lengend Snippet: Fig. 1. Computer model of SA-FMP01458:2 SAPN co-assembly: (A) Model of the FMP014 monomer showing the relative positions of the NANP repeats and the aTSR region and the two CD4 epitopes LIDYNKAALSKFKED and IRHENRMV. (B) A computer generated model of the FMP014D0D1 monomer showing the relative position of the flagellin D0D1 and aTSR regions. (C) Model of an assembled SA-FMP014 containing 58 FMP014 monomers and 2 FMP014D0D1 monomers. (D) Dynamic Light Scatter Plot of SA-FMP01458:2. (E) Electron micrograph of nanoparticles formed in the FMP014DOD1 formulation.

Article Snippet: After 24 h, growth media was removed and replaced with DMEM high glucose (Hyclone, Logan, UT) containing either a SAPN, or recombinant flagellin (Novus Biologicals, Littleton, CO) at the following concentrations: 0.01, 0.1, 1, 10, 100, 1000 ng/mL in triplicate.

Techniques: Generated, Formulation

Journal: Frontiers in immunology

Article Title: Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling.

doi: 10.3389/fimmu.2020.581165

Figure Lengend Snippet: FIGURE 1 | Culture supernatants from Salmonella typhimurium promotes NLRC4-dependent inflammasome activation. (A) Illustration of experimental scheme to determine inflammasome activation in BMDMs by bacteria-secreted molecules using transwell plate. (B) Immunoblots from mouse BMDMs in the lower well (A) incubated with wild-type (WT) or DfliC-fljB S. typhimurium SL1344 in the upper well (A, MOI 30) for 7 h. (C) Immunoblots from mouse BMDMs untreated or treated with culture supernatant (CS, 1/33 or 1/10 volume of culture medium), derived from 6 h culture of WT or DfliC–fljB S. typhimurium, for 6 h. (D) Immunoblots from Nlrc4+/+ or Nlrc4−/−mice BMDMs treated with WT or DfliC–fljB S. typhimurium CS (1/10) for 6 h. (E) Immunoblots from Nlrp3+/+ or Nlrp3−/−mice BMDMs treated with WT or DfliC–fljB S. typhimurium CS (1/10) for 6 h, or primed with LPS (0.25 µg/ml, 3 h), followed by treatment with ATP (2.5 mM, 40 min). (F) Immunoblots from mouse BMDMs incubated with S. typhimurium CS (1/20) with or without heat treatment (97°C, 30 min) or proteinase K treatment (10 µg/ml, 30 min), for 6 h. (G, H) Quantification of IL-1b (G) or IL-6 (H) in the supernatant of mouse BMDMs treated with S. typhimurium CS (1/20) as same as in (F). (n = 3, one-way ANOVA) (I, J) Quantification of IL-1b (I) or IL-6 (J) in the culture supernatants of mouse BMDMs treated with WT, DfliC–fljB, or DfliC–fljB–prgJ S. typhimurium CS (1/100) for 6 h. (n = 4, one-way ANOVA). (K) Immunoblots from mouse BMDMs treated with culture supernatant (CS, 1/100 or 1/20), derived from 6 h culture of WT, DfliC–fljB, DfliC–fljB–prgJ S. typhimurium 14028s, for 6 h. (L) Immunoblots from mouse BMDMs treated with S. typhimurium flagellin (ST-FLA, 250 ng/ml) with or without premixing with DOTAP (DT) liposomal transfection reagent for 6 h or treated with LPS (0.25 µg/ml, 3 h), followed by ATP treatment (2.5 mM, 40 min). Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences compared with S. typhimurium CS-treated group. (*P < 0.05, ***P < 0.001, n.s. not significant).

Article Snippet: The following antibodies were used for detecting caspase-1 (Adipogen), NLRP3 (Adipogen), IL-1b (R&D Systems), ASC (Santa Cruz Biotechnology), flagellin (InvivoGen), OmpA (Biorbyt), and gasdermin D (Novus Biologicals).

Techniques: Activation Assay, Bacteria, Western Blot, Incubation, Derivative Assay, Transfection

Journal: Frontiers in immunology

Article Title: Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling.

doi: 10.3389/fimmu.2020.581165

Figure Lengend Snippet: FIGURE 5 | Salmonella-released outer membrane vesicles exhibit heat-resistant and GBP2-independent inflammasome activation potential. (A) Immunoblots of OptiPrep density gradient fractions of S. typhimurium OMVs. (B) Immunoblots of the extracts of OMVs in the presence of heat treatment (97°C, 30 min) or proteinase K treatment (10 µg/ml, 30 min) by anti-ST-flagellin antibody. (C) Immunoblots from mouse BMDMs primed with LPS (0.25 µg/ml, 3 h), followed by the treatment with intact or heat-treated S. typhimurium flagellin (250 ng/ml) for 6 h. (D) Immunoblots of mouse BMDMs treated with intact or heat-treated S. typhimurium OMVs (5 µg/ml) for 6 h. (E) Quantification of IL-1b in the culture supernatants of mouse BMDMs treated with intact or heat-treated S. typhimurium OMVs for 6 h. (n = 5). (F) Immunoblots from Gbp2+/+ or Gbp2−/−mice BMDMs treated with E. coli BL21 OMVs (5 µg/ml) or S. typhimurium (14028s) OMVs (5 µg/ml) or for 8 h. (G, H) Quantification of IL-1b in the culture supernatants of Gbp2+/+ or Gbp2−/−mice BMDMs treated as same as (F) (n = 3). (I, J) Immunoblots of mouse BMDMs treated with intact or proteinase-treated S. typhimurium OMVs (10 µg/ml) in the presence of MCC 950 (100 nM) as indicated for 8 h. After proteinase treatment, PMSF was added to eliminate proteinase K activity. Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences. (**P < 0.01, ***P < 0.001, n.s. not significant).

Article Snippet: The following antibodies were used for detecting caspase-1 (Adipogen), NLRP3 (Adipogen), IL-1b (R&D Systems), ASC (Santa Cruz Biotechnology), flagellin (InvivoGen), OmpA (Biorbyt), and gasdermin D (Novus Biologicals).

Techniques: Membrane, Activation Assay, Western Blot, Activity Assay

Journal: Frontiers in immunology

Article Title: Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling.

doi: 10.3389/fimmu.2020.581165

Figure Lengend Snippet: FIGURE 7 | Proposed mechanism of host inflammasome activation by flagellated or non-flagellated bacteria-released outer membrane vesicles. Flagellated bacteria such as S. typhimurium or P. aeruginosa facilitate the delivery of bacterial flagellin and LPS into the cytoplasm of host cells via OMV cargo. In the initial phase of OMV exposure, OMV-delivered flagellin causes the assembly of NLRC4 inflammasome leading to caspase-1 activation and the subsequent IL-1b secretion. Besides flagellin, intracellular OMV-associated LPS can activate caspase-11 non-canonical inflammasome signaling, which includes GSDMD-dependent pyroptosis and NLRP3-mediated caspase-1 activation and IL-1b secretion. On the contrary, OMVs derived from non-flagellated bacteria such as E. coli mediates the cytosolic delivery of bacterial LPS. Non-flagellated bacterial -delivered LPS mainly causes the activation of caspase-11 and GSDMD-mediated pyroptosis, contributing to potential endotoxic shock. NAIP5, NLR family, apoptosis inhibitory protein 5; NLRC4, NLR family, CARD domain-containing protein 4; NLRP3, NLR family, pyrin domain-containing protein 3; GSDMD, gasdermin D.

Article Snippet: The following antibodies were used for detecting caspase-1 (Adipogen), NLRP3 (Adipogen), IL-1b (R&D Systems), ASC (Santa Cruz Biotechnology), flagellin (InvivoGen), OmpA (Biorbyt), and gasdermin D (Novus Biologicals).

Techniques: Activation Assay, Bacteria, Membrane, Derivative Assay