mouse tradd  (Novus Biologicals)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Salmonella Effectors SseK1 and SseK3 Target Death Domain Proteins in the TNF and TRAIL Signaling Pathways*

doi: 10.1074/mcp.RA118.001093

Figure Lengend Snippet: Mutagenesis of putative SseK1 glycosylation sites of TRADD. A, Immunoblot showing Arg-GlcNAcylation of ectopically expressed Flag-hTRADD or Flag-hTRADD mutants in HEK293T cells co-transfected with pEGFP-SseK1. Cells were harvested for immunoblotting and detected with anti-ArgGlcNAc, anti-GFP, and anti-Flag antibodies. Antibodies to β-actin were used as a loading control. Representative immunoblot of at least three independent experiments. B, Manually curated EThcD spectra of Arg-GlcNAcylated Flag-hTRADD enriched by anti-Flag immunoprecipitation following ectopic expression in HEK293T cells and co-transfection with pEGFP-SseK1. Various observed sites of Arg-GlcNAcylation are highlighted in red, and presented alongside corresponding M/Z values and observed Andromeda scores. Within MS/MS spectra NL denote neutral loss associated ions.

Article Snippet: Membranes were rinsed and washed in TBS Tween, then probed one of the following primary antibodies as required at 4 °C overnight with shaking at 60 rpm: rabbit monoclonal anti-ArgGlcNAc (Abcam, Cambridge, UK), mouse monoclonal anti-HA (BioLegend, San Diego CA), mouse monoclonal anti-GFP (Roche, Basel, Switzerland), mouse monoclonal anti-Flag M2-HRP (Sigma-Aldrich, St Louis, MO), mouse monoclonal anti-TRADD (BD Biosciences, San Jose, CA) or mouse monoclonal anti-β-actin (Sigma).

Techniques: Mutagenesis, Western Blot, Transfection, Immunoprecipitation, Expressing, Cotransfection, Tandem Mass Spectroscopy

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: CFTR controls the activity of NF-kB by enhancing the degradation of tradd

doi: 10.1159/000453162

Figure Lengend Snippet: 1A) CFBE cells were transfected with GFP-wt CFTR, GFP-ΔF508 CFTR, GFP-TNR CFTR, or GFP-G551D CFTR. Forty-eight hours after transfection, cells were lysed, and co-immunoprecipitation was performed using either anti-TRADD or mouse IgG (immunoglobulin G) antibodies. CFTR expression in the total cell lysates is shown, as well as CFTR, which is co-precipitated with TRADD. The results show that TRADD binds to wt and G551D but not to ΔF508 and TNR CFTR, both of which remain in the ER, suggesting that TRADD binds only to CFTR that is processed to the plasma membrane. (1B-C) CFBE cells were transfected with GFP-wt CFTR (1B) or GFP-ΔF508 CFTR (1C). Antibodies recognizing GFP (green) and TRADD (red) were used in immunofluorescence-based detection. wt CFTR and TRADD were colocalized in the perinuclear region of the cells. Importantly, TRADD did not colocalize with ΔF508 CFTR, consistent with the lack of binding of this mutant. # - denotes that CFBE cells were transfected CFTR.

Article Snippet: We used monoclonal anti-human CFTR antibody 217 (University of North Carolina), mouse anti-TRADD monoclonal antibody (mAb;1:500, BD Transduction Laboratories), and rabbit polyclonal anti-β-actin antibody (1:1000, SantaCruz Biotechnology).

Techniques: Transfection, Immunoprecipitation, Expressing, Immunofluorescence, Binding Assay, Mutagenesis

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: CFTR controls the activity of NF-kB by enhancing the degradation of tradd

doi: 10.1159/000453162

Figure Lengend Snippet: NF-κB was measured in CFBE cells transfected with either wt CFTR or ΔF508 CFTR, with and without knockdown (KD) of TRADD using shRNA. There was no difference in total NF-κB protein between the parental CFBE cells and those stably expressing wt CFTR (6A). In (6B) and summarized in (6C), transfection of wt CFTR into CFBE cells in TRADD-containing cells caused a reduction in NF-κB activity when compared to cells transfected with ΔF508 CFTR. The difference in nuclear NF-κB between wt and ΔF508 CFTR-expressing cells was less in cells in which TRADD levels were reduced by treatment with shRNA. Results are means ± S.E. (n=3). *, p<0.05.

Article Snippet: We used monoclonal anti-human CFTR antibody 217 (University of North Carolina), mouse anti-TRADD monoclonal antibody (mAb;1:500, BD Transduction Laboratories), and rabbit polyclonal anti-β-actin antibody (1:1000, SantaCruz Biotechnology).

Techniques: Transfection, shRNA, Stable Transfection, Expressing, Activity Assay

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: CFTR controls the activity of NF-kB by enhancing the degradation of tradd

doi: 10.1159/000453162

Figure Lengend Snippet: CFBE cells were transfected with GFP-CAL. (2A). TRADD alone. (2B). Cal alone. (2C) is the merged image. Note that CAL colocalized with TRADD and bound to TRADD in co-immunoprecipitation experiments (2D). Since we have shown previously [28] that CAL is localized at the TGN and also binds CFTR via the PDZ domain, these data suggest that TRADD and wt CFTR colocalize in the TGN.

Article Snippet: We used monoclonal anti-human CFTR antibody 217 (University of North Carolina), mouse anti-TRADD monoclonal antibody (mAb;1:500, BD Transduction Laboratories), and rabbit polyclonal anti-β-actin antibody (1:1000, SantaCruz Biotechnology).

Techniques: Transfection, Immunoprecipitation

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: CFTR controls the activity of NF-kB by enhancing the degradation of tradd

doi: 10.1159/000453162

Figure Lengend Snippet: Parental CFBE cells and CFBE stably expressing wt CFTR were transfected with either TRADD or a control vector. (3A) CFTR was detected by Western blotting using an anti-CFTR monoclonal antibody. Note that there is much less endogenous CFTR in the untransfected CFBE cells compared to those stably expressing wt-CFTR. Thus, in this western blot containing wt-CFTR the endogenous ΔF508 CFTR is not evident. β-actin was used as a loading control. TRADD overexpression dramatically increased the expression of mature C-band of wt CFTR but had no effect on the immature B-band, nor did it rescue ΔF508 CFTR. (3B) Results are means ± S.E. *, p<0.05 versus the control. (n=3), Data was normalized to CFBE-WT CFTR TRADD transfected.

Article Snippet: We used monoclonal anti-human CFTR antibody 217 (University of North Carolina), mouse anti-TRADD monoclonal antibody (mAb;1:500, BD Transduction Laboratories), and rabbit polyclonal anti-β-actin antibody (1:1000, SantaCruz Biotechnology).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Western Blot, Over Expression

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: CFTR controls the activity of NF-kB by enhancing the degradation of tradd

doi: 10.1159/000453162

Figure Lengend Snippet: Parental CFBE cells and CFBE stably expressing wt CFTR were transfected with either TRADD or a control vector. (4A) Cell-surface CFTR was detected using a cell-surface biotinylation assay. CFTR was detected by Western blotting using an anti-CFTR monoclonal antibody. β-actin was used as a loading control. TRADD overexpression increased the cell-surface expression of wt CFTR but had no effect on ΔF508 CFTR. Note that there is no surface expression of ΔF508 CFTR in the parental CFBE cells as expected because this mutant does not reach the plasma membrane. (4B) Results are means ± S.E. *, p<0.05 ) (n=3).versus the control.

Article Snippet: We used monoclonal anti-human CFTR antibody 217 (University of North Carolina), mouse anti-TRADD monoclonal antibody (mAb;1:500, BD Transduction Laboratories), and rabbit polyclonal anti-β-actin antibody (1:1000, SantaCruz Biotechnology).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Cell Surface Biotinylation Assay, Western Blot, Over Expression, Mutagenesis

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: CFTR controls the activity of NF-kB by enhancing the degradation of tradd

doi: 10.1159/000453162

Figure Lengend Snippet: There was no difference in the total NF-κB protein in either the parental CFBE cells or those stably expressing wt CFTR (5A). In (5B) and summarized in (5C), NF-κB activity was measured with an NF-κB assay kit (see Methods) using an antibody against the p65 (RelA) NF-κB transcription factor subunit. The assay takes advantage of the process of activation of NF-κB. When activated, the IkB inhibitory subunit is degraded, allowing the transcription factor subunits of the complex to translocate to the nucleus [65]. In the assay, p65 reactivity was measured in the nuclear extracts by detecting the chemiluminescence of the sample using FujiFilm LAS-3000. Since there was no change in total NF-kB, the p65 reactivity was measured in the nuclear extract and compared across the experimental maneuvers. Results are normalized to control. Results are means ± S.E. (n=3). *, p<0.05. Transfection of TRADD increased NF-kB activity only in CFBE cells containing ΔF508 CFTR, and not in cells expressing wt CFTR. NF-κB was measured in CFBE cells and CFBE stably expressing wt CFTR with and without the transfection of additional TRADD.

Article Snippet: We used monoclonal anti-human CFTR antibody 217 (University of North Carolina), mouse anti-TRADD monoclonal antibody (mAb;1:500, BD Transduction Laboratories), and rabbit polyclonal anti-β-actin antibody (1:1000, SantaCruz Biotechnology).

Techniques: Stable Transfection, Expressing, Activity Assay, Activation Assay, Transfection

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: CFTR controls the activity of NF-kB by enhancing the degradation of tradd

doi: 10.1159/000453162

Figure Lengend Snippet: CFBE-wt CFTR, CFBE, and CFBE cells transfected with G551D CFTR were treated with 10 μM CFTRinh172, 10 μM forskolin, or 100 μM IBMX for 4 h as specified in the figure. (7A) CFTRinh172 significantly increased NF-κB in the nucleus only in CFBE wt CFTR cells, and not in CFBE or CFBE cells transfected with G551D. TBP served as a nuclear loading control. (7B). Results are means ± S.E (n=3). *, p<0.05. (7 C-D) In CFBE cells, TRADD was co-transfected with GFP-wt CFTR, GFP-ΔF508 CFTR, GFP-TNR CFTR, S341A CFTR, or G551D CFTR. NF-kB activity was reduced by wt CFTR, but not by any of the other tested mutations of CFTR. TBP served as a nuclear loading control. (7D). Results are means ± S.E (n=3).

Article Snippet: We used monoclonal anti-human CFTR antibody 217 (University of North Carolina), mouse anti-TRADD monoclonal antibody (mAb;1:500, BD Transduction Laboratories), and rabbit polyclonal anti-β-actin antibody (1:1000, SantaCruz Biotechnology).

Techniques: Transfection, Activity Assay

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: CFTR controls the activity of NF-kB by enhancing the degradation of tradd

doi: 10.1159/000453162

Figure Lengend Snippet: CFBE wt CFTR cells and CFBE cells were transfected with shRNA to knock down TRADD. The cells were treated with 10 μM CFTRinh172, 10 μM forskolin, or 100 μM IBMX for 4 h as specified in the figure. (8A) When TRADD was knocked down, CFTRinh172 did not change NF-κB located in the nucleus (n=3); *, p<0.05). TBP served as a nuclear loading control. (8B). Results are means ± S.E.

Article Snippet: We used monoclonal anti-human CFTR antibody 217 (University of North Carolina), mouse anti-TRADD monoclonal antibody (mAb;1:500, BD Transduction Laboratories), and rabbit polyclonal anti-β-actin antibody (1:1000, SantaCruz Biotechnology).

Techniques: Transfection, shRNA

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: CFTR controls the activity of NF-kB by enhancing the degradation of tradd

doi: 10.1159/000453162

Figure Lengend Snippet: (10 A-B) In CFBE cells, TRADD was co-transfected with GFP-wt CFTR, GFP-ΔF508, or GFP- G551D. WT-CFTR transfected cells were treated with vehicle or CFTRinh172. TRADD protein expression was increased when cells containing wt CFTR. (10 C-D) In CFBE cells, TRADD was co-transfected with GFP-wt CFTR, GFP-ΔF508, or GFP-ΔTRL CFTR. At 24 h after transfection, cells were treated with proteasome inhibitor (MG-132) or lysosome inhibitor (E64) for 16 h. MG132 and E64 treatment significantly increased TRADD protein expression only when wt CFTR was expressed. (10 D) Results are means ± S.E. *, p<0.05 (n=3).

Article Snippet: We used monoclonal anti-human CFTR antibody 217 (University of North Carolina), mouse anti-TRADD monoclonal antibody (mAb;1:500, BD Transduction Laboratories), and rabbit polyclonal anti-β-actin antibody (1:1000, SantaCruz Biotechnology).

Techniques: Transfection, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: CFTR controls the activity of NF-kB by enhancing the degradation of tradd

doi: 10.1159/000453162

Figure Lengend Snippet: (9A) TRADD expression was significantly reduced after shRNA transfection. 30ng/ml TNF treatment increased NF-kB levels in CFBE-wt CFTR and CFBE cells, and this enhancement was most notable in CFBE cells containing TRADD. (9B) Results are means ± S.E. *, p<0.05 (n=3).

Article Snippet: We used monoclonal anti-human CFTR antibody 217 (University of North Carolina), mouse anti-TRADD monoclonal antibody (mAb;1:500, BD Transduction Laboratories), and rabbit polyclonal anti-β-actin antibody (1:1000, SantaCruz Biotechnology).

Techniques: Expressing, shRNA, Transfection

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: CFTR controls the activity of NF-kB by enhancing the degradation of tradd

doi: 10.1159/000453162

Figure Lengend Snippet: (11A -B) Parental CFBE and cells stably expressing wt-CFTR were treated with proteasome inhibitor (MG-132) for 16 hours prior to exposure to cycloheximide to block protein translation. (11C-D) Parental CFBE and cells stably expressing wt-CFTR were treated with bafilomycin [66]which prevents degradation of proteins by the lysosome for 16 hours prior to exposure to cycloheximide to block protein translation[67]. Note that in the presence of CFTR that the disappearance of TRADD is greater than in the absence of CFTR when the proteasome is blocked. When the lysosome is blocked the expression of TRADD protein is stable over the experimental period indicating that CFTR enhances the degradation of TRADD by the lysosome. Results are means ± S.E. *, p<0.05 (n=3).

Article Snippet: We used monoclonal anti-human CFTR antibody 217 (University of North Carolina), mouse anti-TRADD monoclonal antibody (mAb;1:500, BD Transduction Laboratories), and rabbit polyclonal anti-β-actin antibody (1:1000, SantaCruz Biotechnology).

Techniques: Stable Transfection, Expressing, Blocking Assay

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: CFTR controls the activity of NF-kB by enhancing the degradation of tradd

doi: 10.1159/000453162

Figure Lengend Snippet: 12A. TNFα forms a complex with TNFR, which binds to its obligate intracellular adaptor TRADD. The TRADD complex drives proinflammatory signaling by activating NFκB. Wt CFTR inhibits TRADD by inducing degradation to the lysosome. The result is suppression of NFκB activation. ShRNA against TRADD reduces TRADD and blocks TNFα-activated NFκB activation. Elevation of TRADD expression feeds back to elevate Wt CFTR. CFTRinh172 blocks channel activity by Wt CFTR, and blocks inhibitor of TRADD by Wt CFTR. Color code: red = activation; green = inhibition. 12B). In the presence of mutant ΔF508 CFTR, TRADD is not inhibited. TNFα/TNFR (R=receptor) can now bind to TRADD, and drive activation of NFκB. The “*” signs indicate “activation” of the TNFα/TNFR complex and the NFkB complexes, respectively.

Article Snippet: We used monoclonal anti-human CFTR antibody 217 (University of North Carolina), mouse anti-TRADD monoclonal antibody (mAb;1:500, BD Transduction Laboratories), and rabbit polyclonal anti-β-actin antibody (1:1000, SantaCruz Biotechnology).

Techniques: Activation Assay, shRNA, Expressing, Activity Assay, Inhibition, Mutagenesis