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    Proteintech rabbit ing2 polyclonal
    MRPL12 mediated the effects of <t>ING2</t> on mitochondrial OXPHOS in tubular epithelial cells. (A,B) HK2 cells were transfected with ING2-shRNA or ING2-overexpression plasmid. The mRNA and protein levels of TFAM and MRPL12 were evaluated by qPCR and Western blotting. (C,D) HK2 cells were divided into control group, ING2 overexpression group, MRPL12 knockout group, and MRPL12 knockout plus ING2 overexpression group. Mitochondrial OXPHOS was detected using Seahorse. OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; A R, antimycin and rotenone. (E) Western blotting to validate the altered expression of mtDNA-encoded components of mitochondria complex. Data were from three individual experiments and presented as mean ± SEM. * p
    Rabbit Ing2 Polyclonal, supplied by Proteintech, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher anti ddx3 shrnas
    Protein synthesis in BAdV-3 infected DDX3kd VIDOR2 cells. (A) Western blot of lysates of <t>DDX3</t> +Ve or DDX3kd VIDOR2 cells with anti-DDX3 antibody. (B,C) [ 35 S] methionine pulse labeling and quantification. Mock infected or BAdV-3 infected cells were pulse labeled with [ 35 S] methionine for 10 min at 36 h or 48 h post infection. The radiolabelled proteins were separated by 10% SDS-PAGE and analyzed by autoradiography. (C) The intensity of each lane was quantified using GelQuant.NET software and plotted relative to the intensity of the mock infected cell lanes.
    Anti Ddx3 Shrnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ddx3 shrnas/product/Thermo Fisher
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ddx3 shrnas - by Bioz Stars, 2022-07
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    Image Search Results


    MRPL12 mediated the effects of ING2 on mitochondrial OXPHOS in tubular epithelial cells. (A,B) HK2 cells were transfected with ING2-shRNA or ING2-overexpression plasmid. The mRNA and protein levels of TFAM and MRPL12 were evaluated by qPCR and Western blotting. (C,D) HK2 cells were divided into control group, ING2 overexpression group, MRPL12 knockout group, and MRPL12 knockout plus ING2 overexpression group. Mitochondrial OXPHOS was detected using Seahorse. OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; A R, antimycin and rotenone. (E) Western blotting to validate the altered expression of mtDNA-encoded components of mitochondria complex. Data were from three individual experiments and presented as mean ± SEM. * p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

    doi: 10.3389/fcell.2021.700195

    Figure Lengend Snippet: MRPL12 mediated the effects of ING2 on mitochondrial OXPHOS in tubular epithelial cells. (A,B) HK2 cells were transfected with ING2-shRNA or ING2-overexpression plasmid. The mRNA and protein levels of TFAM and MRPL12 were evaluated by qPCR and Western blotting. (C,D) HK2 cells were divided into control group, ING2 overexpression group, MRPL12 knockout group, and MRPL12 knockout plus ING2 overexpression group. Mitochondrial OXPHOS was detected using Seahorse. OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; A R, antimycin and rotenone. (E) Western blotting to validate the altered expression of mtDNA-encoded components of mitochondria complex. Data were from three individual experiments and presented as mean ± SEM. * p

    Article Snippet: Cells were labeled with antibodies against ING2 (Proteintech, 11560-1-AP, 1:200) or MRPL12 (Proteintech, 14795-1-AP, 1:200) at 4°C overnight.

    Techniques: Transfection, shRNA, Over Expression, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Knock-Out, Expressing

    ING2 positively regulates mitochondrial respiration in tubular epithelial cells. HK2 cells were transfected with ING2-shRNA or ING2-overexpression plasmid. (A,B) The transfection efficiencies were evaluated by qPCR and Western blotting, respectively. (C–F) Following transfection, mitochondrial OXPHOS of HK2 cells were detected using Seahorse. OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; A R, antimycin and rotenone. (G,H) mtDNA-encoded components for complex I, III, IV, and V and 16S rRNA were determined by qPCR. (I) mtDNA-encoded components for complex I, III, IV, and V were determined by Western blotting. (J) The mitochondria were stained with MitoTracker (red) and ING2 (green) followed by DAPI (blue) re-dyeing. (K) The mitochondria DNA (mtDNA) copy number was determined by qPCR with G6PC serving as internal reference. Data were from three individual experiments and presented as mean ± SEM. * p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

    doi: 10.3389/fcell.2021.700195

    Figure Lengend Snippet: ING2 positively regulates mitochondrial respiration in tubular epithelial cells. HK2 cells were transfected with ING2-shRNA or ING2-overexpression plasmid. (A,B) The transfection efficiencies were evaluated by qPCR and Western blotting, respectively. (C–F) Following transfection, mitochondrial OXPHOS of HK2 cells were detected using Seahorse. OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; A R, antimycin and rotenone. (G,H) mtDNA-encoded components for complex I, III, IV, and V and 16S rRNA were determined by qPCR. (I) mtDNA-encoded components for complex I, III, IV, and V were determined by Western blotting. (J) The mitochondria were stained with MitoTracker (red) and ING2 (green) followed by DAPI (blue) re-dyeing. (K) The mitochondria DNA (mtDNA) copy number was determined by qPCR with G6PC serving as internal reference. Data were from three individual experiments and presented as mean ± SEM. * p

    Article Snippet: Cells were labeled with antibodies against ING2 (Proteintech, 11560-1-AP, 1:200) or MRPL12 (Proteintech, 14795-1-AP, 1:200) at 4°C overnight.

    Techniques: Transfection, shRNA, Over Expression, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Staining

    ING2 inhibited the ubiquitination of MRPL12. (A) HK2 cells were transfected with control or ING2-shRNA, followed by MG132 treatment for 6 h. Protein levels of ING2 and MRPL12 were evaluated by Western blotting. (B) A ubiquitin modified site was found at 58 lysine residue (red, upper panel), predicted using predictor of protein ubiquitination sites, UbPred. (C,D) The ubiquitination of MRPL12 was determined by proximity ligation assay (PLA) and immunoprecipitation, respectively, after transfected ING2-overexpression plasmid. (E) By using ASEB, a web tool for predicting protein acetylation site, a acetylation site was detected at the 185 lysine residue (green, lower panel). (F) The acetylation of MRPL12 was determined by immunoprecipitation after transfected ING2-overexpression plasmid. IP, immunoprecipitation; WB, Western blotting. Data were from three individual experiments.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

    doi: 10.3389/fcell.2021.700195

    Figure Lengend Snippet: ING2 inhibited the ubiquitination of MRPL12. (A) HK2 cells were transfected with control or ING2-shRNA, followed by MG132 treatment for 6 h. Protein levels of ING2 and MRPL12 were evaluated by Western blotting. (B) A ubiquitin modified site was found at 58 lysine residue (red, upper panel), predicted using predictor of protein ubiquitination sites, UbPred. (C,D) The ubiquitination of MRPL12 was determined by proximity ligation assay (PLA) and immunoprecipitation, respectively, after transfected ING2-overexpression plasmid. (E) By using ASEB, a web tool for predicting protein acetylation site, a acetylation site was detected at the 185 lysine residue (green, lower panel). (F) The acetylation of MRPL12 was determined by immunoprecipitation after transfected ING2-overexpression plasmid. IP, immunoprecipitation; WB, Western blotting. Data were from three individual experiments.

    Article Snippet: Cells were labeled with antibodies against ING2 (Proteintech, 11560-1-AP, 1:200) or MRPL12 (Proteintech, 14795-1-AP, 1:200) at 4°C overnight.

    Techniques: Transfection, shRNA, Western Blot, Modification, Proximity Ligation Assay, Immunoprecipitation, Over Expression, Plasmid Preparation

    ING2 ameliorated the ischemia induced mitochondrial OXPHOS defects and tubular cell apoptosis. (A) Immunostaining of kidney tissue section from healthy individual and AKI patients with antibodies against ING2. At least two patients for each. (B) Immunostaining of kidney tissue section from healthy and AKI mice with antibodies against ING2 and MRPL12. HK2 cells were subjected to serum deprivation for 48 h, and protein contents of both ING2, MRPL12, and mtDNA-encoded components of mitochondria complex (C) were figured out by Western blotting. The apoptosis of HK2 cells was evaluated by flow cytometry (D) . HK2 cells were divided into control group, ING2 overexpression group, serum deprivation group, and serum deprivation plus ING2 overexpression group. MRPL12 and mtDNA-encoded components of mitochondria complex were detected through Western blotting (E) . Mitochondrial OXPHOS was detected using Seahorse (F,G) . The apoptosis of HK2 cells was evaluated by flow cytometry (H) , and the ubiquitination of MRPL12 was determined by proximity ligation assay (PLA) (I) . OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; A R, antimycin and rotenone. Data were from three individual experiments and presented as mean ± SEM. * p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

    doi: 10.3389/fcell.2021.700195

    Figure Lengend Snippet: ING2 ameliorated the ischemia induced mitochondrial OXPHOS defects and tubular cell apoptosis. (A) Immunostaining of kidney tissue section from healthy individual and AKI patients with antibodies against ING2. At least two patients for each. (B) Immunostaining of kidney tissue section from healthy and AKI mice with antibodies against ING2 and MRPL12. HK2 cells were subjected to serum deprivation for 48 h, and protein contents of both ING2, MRPL12, and mtDNA-encoded components of mitochondria complex (C) were figured out by Western blotting. The apoptosis of HK2 cells was evaluated by flow cytometry (D) . HK2 cells were divided into control group, ING2 overexpression group, serum deprivation group, and serum deprivation plus ING2 overexpression group. MRPL12 and mtDNA-encoded components of mitochondria complex were detected through Western blotting (E) . Mitochondrial OXPHOS was detected using Seahorse (F,G) . The apoptosis of HK2 cells was evaluated by flow cytometry (H) , and the ubiquitination of MRPL12 was determined by proximity ligation assay (PLA) (I) . OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; A R, antimycin and rotenone. Data were from three individual experiments and presented as mean ± SEM. * p

    Article Snippet: Cells were labeled with antibodies against ING2 (Proteintech, 11560-1-AP, 1:200) or MRPL12 (Proteintech, 14795-1-AP, 1:200) at 4°C overnight.

    Techniques: Immunostaining, Mouse Assay, Western Blot, Flow Cytometry, Over Expression, Proximity Ligation Assay

    ING2 overexpression effectively ameliorated ischemic kidney injury. The mice were injected with rAAV-con or rAAV-ING2 followed by the induction of IRI with sham operation group as control. (A) Immunofluorescent staining of kidneys after the intra-renal injection of rAAV vectors with ING2 (red) and DAPI (blue). (B) Immunostaining of renal tissue sections with antibodies against ING2, MRPL12, ND2, and COX II. (C) Western blot analysis of renal tissue ING2, MRPL12, ND2, and COX II. (D) Levels of creatinine in the serum or urine of IRI and sham operation mice. Data were from two individual experiments and presented as mean ± SEM. rAAV-con + IRI, n = 6; rAAV-ING2 + IRI, n = 6; rAAV-con + sham, n = 4; rAAV-ING2 + sham, n = 4. ** p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

    doi: 10.3389/fcell.2021.700195

    Figure Lengend Snippet: ING2 overexpression effectively ameliorated ischemic kidney injury. The mice were injected with rAAV-con or rAAV-ING2 followed by the induction of IRI with sham operation group as control. (A) Immunofluorescent staining of kidneys after the intra-renal injection of rAAV vectors with ING2 (red) and DAPI (blue). (B) Immunostaining of renal tissue sections with antibodies against ING2, MRPL12, ND2, and COX II. (C) Western blot analysis of renal tissue ING2, MRPL12, ND2, and COX II. (D) Levels of creatinine in the serum or urine of IRI and sham operation mice. Data were from two individual experiments and presented as mean ± SEM. rAAV-con + IRI, n = 6; rAAV-ING2 + IRI, n = 6; rAAV-con + sham, n = 4; rAAV-ING2 + sham, n = 4. ** p

    Article Snippet: Cells were labeled with antibodies against ING2 (Proteintech, 11560-1-AP, 1:200) or MRPL12 (Proteintech, 14795-1-AP, 1:200) at 4°C overnight.

    Techniques: Over Expression, Mouse Assay, Injection, Staining, Immunostaining, Western Blot

    Protein synthesis in BAdV-3 infected DDX3kd VIDOR2 cells. (A) Western blot of lysates of DDX3 +Ve or DDX3kd VIDOR2 cells with anti-DDX3 antibody. (B,C) [ 35 S] methionine pulse labeling and quantification. Mock infected or BAdV-3 infected cells were pulse labeled with [ 35 S] methionine for 10 min at 36 h or 48 h post infection. The radiolabelled proteins were separated by 10% SDS-PAGE and analyzed by autoradiography. (C) The intensity of each lane was quantified using GelQuant.NET software and plotted relative to the intensity of the mock infected cell lanes.

    Journal: Frontiers in Microbiology

    Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

    doi: 10.3389/fmicb.2016.02119

    Figure Lengend Snippet: Protein synthesis in BAdV-3 infected DDX3kd VIDOR2 cells. (A) Western blot of lysates of DDX3 +Ve or DDX3kd VIDOR2 cells with anti-DDX3 antibody. (B,C) [ 35 S] methionine pulse labeling and quantification. Mock infected or BAdV-3 infected cells were pulse labeled with [ 35 S] methionine for 10 min at 36 h or 48 h post infection. The radiolabelled proteins were separated by 10% SDS-PAGE and analyzed by autoradiography. (C) The intensity of each lane was quantified using GelQuant.NET software and plotted relative to the intensity of the mock infected cell lanes.

    Article Snippet: Proteins were separated by 10%SDS-PAGE gel, transferred to nitrocellulose and probed in Western blot using anti-DDX3 or anti-eIF specific antibody followed by Alexa Flour 680 goat anti-rabbit IgG (Molecular Probes) or IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) as secondary antibody.

    Techniques: Infection, Western Blot, Labeling, SDS Page, Autoradiography, Software

    Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).

    Journal: Frontiers in Microbiology

    Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

    doi: 10.3389/fmicb.2016.02119

    Figure Lengend Snippet: Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).

    Article Snippet: Proteins were separated by 10%SDS-PAGE gel, transferred to nitrocellulose and probed in Western blot using anti-DDX3 or anti-eIF specific antibody followed by Alexa Flour 680 goat anti-rabbit IgG (Molecular Probes) or IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) as secondary antibody.

    Techniques: Staining, Purification, SDS Page, Pull Down Assay, Incubation, In Vitro, Labeling, Autoradiography, Immunoprecipitation

    Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.

    Journal: Frontiers in Microbiology

    Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

    doi: 10.3389/fmicb.2016.02119

    Figure Lengend Snippet: Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.

    Article Snippet: Proteins were separated by 10%SDS-PAGE gel, transferred to nitrocellulose and probed in Western blot using anti-DDX3 or anti-eIF specific antibody followed by Alexa Flour 680 goat anti-rabbit IgG (Molecular Probes) or IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) as secondary antibody.

    Techniques: Pull Down Assay, Purification, Incubation, In Vitro, Labeling, SDS Page, Autoradiography, Immunoprecipitation, Transfection, Western Blot, Infection, CTL Assay, Confocal Microscopy, Plasmid Preparation, Immunofluorescence, Fluorescence, Staining

    Interaction of pVIII with eIFs. (A) The cytoplasmic fraction (50 μl) of DDX3 positive or negative VIDO R2 cells were incubated with 10 μl of beads loaded with 750 ng of purified GST-VIII protein for 2 h at 4°C and centrifuged for 10 min. The supernatant (S) and the pellet (P) from both cytoplasmic fraction were separated by 10% SDS-PAGE gel and analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. Total amount of indicated protein in cytoplasmic fraction of indicated cells was estimated by Western blot analysis before adding GST.pVIII fusion protein. (B) DDX3 knockdown 293T cells were transfected with 2 μg of pHA.DX3 or pHA.pVIII or pHA.DX3 and pc.pVIII. The cells were lysed at 48 h post transfection and precipitated by anti-HA affinity matrix and the precipitates were analyzed by Western blot as indicated above.

    Journal: Frontiers in Microbiology

    Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

    doi: 10.3389/fmicb.2016.02119

    Figure Lengend Snippet: Interaction of pVIII with eIFs. (A) The cytoplasmic fraction (50 μl) of DDX3 positive or negative VIDO R2 cells were incubated with 10 μl of beads loaded with 750 ng of purified GST-VIII protein for 2 h at 4°C and centrifuged for 10 min. The supernatant (S) and the pellet (P) from both cytoplasmic fraction were separated by 10% SDS-PAGE gel and analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. Total amount of indicated protein in cytoplasmic fraction of indicated cells was estimated by Western blot analysis before adding GST.pVIII fusion protein. (B) DDX3 knockdown 293T cells were transfected with 2 μg of pHA.DX3 or pHA.pVIII or pHA.DX3 and pc.pVIII. The cells were lysed at 48 h post transfection and precipitated by anti-HA affinity matrix and the precipitates were analyzed by Western blot as indicated above.

    Article Snippet: Proteins were separated by 10%SDS-PAGE gel, transferred to nitrocellulose and probed in Western blot using anti-DDX3 or anti-eIF specific antibody followed by Alexa Flour 680 goat anti-rabbit IgG (Molecular Probes) or IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) as secondary antibody.

    Techniques: Incubation, Purification, SDS Page, Western Blot, Transfection