curve fits and comparisons (f test) performed with graphpad prism version 4.02 Search Results


graphpad prism software  (GraphPad Software Inc)
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GraphPad Software Inc graphpad prism software
Graphpad Prism Software, supplied by GraphPad Software Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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scion image beta version 4.02  (scion corporation)
90
scion corporation scion image beta version 4.02
Scion Image Beta Version 4.02, supplied by scion corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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antibody anti ubiquitin  (Santa Cruz Biotechnology)
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Santa Cruz Biotechnology antibody anti ubiquitin
Figure 1. Deregulation of canonical Wnt signalling and proteasome activity following loss or mutation of MKS1. (a) Immunoblots for total soluble β-catenin, phospho-β-catenin, cyclin D1 and β-actin (loading control) in either wild-type normal or MKS1-mutated immortalised human fibroblasts from an MKS patient (MKS-562) following treatment with MG-132 proteasome inhibitor (+) or vehicle control (-). (b) SUPER-TOPFlash assays of canonical Wnt signalling activity in human MKS1-mutated fibroblasts compared to wild-type control fibroblasts following treatment with control conditioned medium, Wnt5a, Wnt3a, or a mixture of Wnt3a and Wnt5a media, as indicated. Statistical significance of pairwise comparisons is shown (* indicates p < 0.05, paired two-tailed Student t-test). Error bars indicate s.e.m. with results shown for four independent biological replicates. (c) Proteasome activity assays for wild-type or MKS1-mutated human fibroblasts or an irrelevant control (ASPM-mutant fibroblasts), following treatment with c-lactacystin-β-lactone (+) or vehicle control (-). Statistical significance of pairwise comparison as for (b); *** indicates p < 0.001 for three independent biological replicates. Immunoblots show levels of the 20 S proteasome α7 subunit compared to β-actin loading control. (d) Protease activity assays of crude proteasome preparations from Mks1+/+ or Mks1-/- mouse embryonic fibroblasts (MEFs), expressed as pmol AMC released per µg proteasome per hr. Treatment with lactacystin is the assay control. Statistical analysis as for (b); ** indicates p < 0.01 for three independent biological replicates. (e) Accumulation of GFP-tagged <t>ubiquitin</t> (GFP; green) in Mks1-/- x UbG76V-GFP E12.5 embryonic cerebral neocortex treated with MG-262 proteasome inhibitor. Immunoblot for GFP in Mks1-/- x UbG76V-GFP and wild-type littermate E12.5 embryo protein lysates, with immunoblotting for β-actin as a loading control, showing accumulation of GFP-tagged ubiquitin (Ub-GFP) in Mks1-/-.
Antibody Anti Ubiquitin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Figure 1. Deregulation of canonical Wnt signalling and proteasome activity following loss or mutation of MKS1. (a) Immunoblots for total soluble β-catenin, phospho-β-catenin, cyclin D1 and β-actin (loading control) in either wild-type normal or MKS1-mutated immortalised human fibroblasts from an MKS patient (MKS-562) following treatment with MG-132 proteasome inhibitor (+) or vehicle control (-). (b) SUPER-TOPFlash assays of canonical Wnt signalling activity in human MKS1-mutated fibroblasts compared to wild-type control fibroblasts following treatment with control conditioned medium, Wnt5a, Wnt3a, or a mixture of Wnt3a and Wnt5a media, as indicated. Statistical significance of pairwise comparisons is shown (* indicates p < 0.05, paired two-tailed Student t-test). Error bars indicate s.e.m. with results shown for four independent biological replicates. (c) Proteasome activity assays for wild-type or MKS1-mutated human fibroblasts or an irrelevant control (ASPM-mutant fibroblasts), following treatment with c-lactacystin-β-lactone (+) or vehicle control (-). Statistical significance of pairwise comparison as for (b); *** indicates p < 0.001 for three independent biological replicates. Immunoblots show levels of the 20 S proteasome α7 subunit compared to β-actin loading control. (d) Protease activity assays of crude proteasome preparations from Mks1+/+ or Mks1-/- mouse embryonic fibroblasts (MEFs), expressed as pmol AMC released per µg proteasome per hr. Treatment with lactacystin is the assay control. Statistical analysis as for (b); ** indicates p < 0.01 for three independent biological replicates. (e) Accumulation of GFP-tagged ubiquitin (GFP; green) in Mks1-/- x UbG76V-GFP E12.5 embryonic cerebral neocortex treated with MG-262 proteasome inhibitor. Immunoblot for GFP in Mks1-/- x UbG76V-GFP and wild-type littermate E12.5 embryo protein lysates, with immunoblotting for β-actin as a loading control, showing accumulation of GFP-tagged ubiquitin (Ub-GFP) in Mks1-/-.

Journal: eLife

Article Title: Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

doi: 10.7554/elife.57593

Figure Lengend Snippet: Figure 1. Deregulation of canonical Wnt signalling and proteasome activity following loss or mutation of MKS1. (a) Immunoblots for total soluble β-catenin, phospho-β-catenin, cyclin D1 and β-actin (loading control) in either wild-type normal or MKS1-mutated immortalised human fibroblasts from an MKS patient (MKS-562) following treatment with MG-132 proteasome inhibitor (+) or vehicle control (-). (b) SUPER-TOPFlash assays of canonical Wnt signalling activity in human MKS1-mutated fibroblasts compared to wild-type control fibroblasts following treatment with control conditioned medium, Wnt5a, Wnt3a, or a mixture of Wnt3a and Wnt5a media, as indicated. Statistical significance of pairwise comparisons is shown (* indicates p < 0.05, paired two-tailed Student t-test). Error bars indicate s.e.m. with results shown for four independent biological replicates. (c) Proteasome activity assays for wild-type or MKS1-mutated human fibroblasts or an irrelevant control (ASPM-mutant fibroblasts), following treatment with c-lactacystin-β-lactone (+) or vehicle control (-). Statistical significance of pairwise comparison as for (b); *** indicates p < 0.001 for three independent biological replicates. Immunoblots show levels of the 20 S proteasome α7 subunit compared to β-actin loading control. (d) Protease activity assays of crude proteasome preparations from Mks1+/+ or Mks1-/- mouse embryonic fibroblasts (MEFs), expressed as pmol AMC released per µg proteasome per hr. Treatment with lactacystin is the assay control. Statistical analysis as for (b); ** indicates p < 0.01 for three independent biological replicates. (e) Accumulation of GFP-tagged ubiquitin (GFP; green) in Mks1-/- x UbG76V-GFP E12.5 embryonic cerebral neocortex treated with MG-262 proteasome inhibitor. Immunoblot for GFP in Mks1-/- x UbG76V-GFP and wild-type littermate E12.5 embryo protein lysates, with immunoblotting for β-actin as a loading control, showing accumulation of GFP-tagged ubiquitin (Ub-GFP) in Mks1-/-.

Article Snippet: DOI: https://doi.org/10.7554/eLife.57593 16 of 24 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- mono- and polyubiquitinylated conjugates, clone FK2 (mouse monoclonal) Enzo Life Sciences, Inc ENZ- ABS840 RRID:AB_10541840 WB: 1:1,000 Antibody Anti- 20S proteasome α7 subunit, clone MCP72 (rabbit monoclonal) Enzo Life Sciences Inc BML- PW8110 RRID:AB_10538395 WB: 1:1,000 IF: 1:100 Antibody Anti- MKS1 (rabbit polyclonal) Dawe et al., 2007; Näthke et al., 1996 WB: 1:500 IF: 1:100 Antibody Anti MKS1 (rabbit polyclonal) Proteintech 16206–1- AP RRID:AB_10637856 WB: 1:500 IF: 1:100 Antibody Anti- ubiquitin, clone P4D1 (mouse monoclonal) Santa Cruz Biotechnology, Inc sc- 8017 RRID:AB_2762364 Peptide, recombinant protein MKS1- HIS Proteintech Group, Inc Ag9504 Peptide, recombinant protein UBE2E1- HIS Enzo Life Sciences, Inc UW8710 Peptide, recombinant protein RNF34- HIS Novus Biologicals NBP2- 23440 Peptide, recombinant protein β-catenin- GST Abcam Ab63175 Commercial assay or kit 20 S fluorophore substrate Suc- LLVYAMC Enzo Life Sciences Inc BML- P802- 0005 Commercial assay or kit Dual- Luciferase Reporter Assay system Promega Corp. E1910 Commercial assay or kit Ubiquitination kit Enzo Life Sciences, Inc BML- UW0400 Commercial assay or kit TUBE assays LifeSensors, Malvern, PA, USA UM- 402 Chemical compound, drug MG- 132 Sigma- Aldrich Co. Ltd. C2211 treatment at 10 μM for 3 hr Software, algorithm Prism7 GraphPad Software Inc Continued Informed consent for use of patients in research Informed consent was obtained from all participating families or patients, with studies approved by the Leeds (East) Research Ethics Committee (REC no. 08 /H1306/85) on 4th July 2008.

Techniques: Activity Assay, Mutagenesis, Western Blot, Control, Two Tailed Test, Comparison, Ubiquitin Proteomics

Figure 2. The E2 ubiquitin conjugation enzyme UBE2E1 interacts with MKS1. (a) Domain structure of MKS1 and UBE2E1 proteins for the indicated isoform showing the locations of the B9/C2 domain, putative ubiquitinated lysines in blue (predicted by UbPred), a predicted coiled-coil (CC) motif, and the E2 ubiquitin (UBQ) conjugation domain in UBE2E1. Numbering indicates the amino acid residue. Dashed lines indicate the region used as ‘bait’ in MKS1 for the yeast two-hybrid assay and the ‘prey’ clones in the UBE2E1 interactant. (b) List of preys identified in the MKS1 Y2H screen (c) Left panel: yeast ‘one-to-one’ assays for the indicated bait, prey and control constructs. Right panel: only colonies for the positive control (p53+ SV40 large T) and MKS1 bait+ UBE2E1 prey grew on triple dropout (-Leu -Trp -His) medium. (d) GST-UBE2E1 purified from bacterial extracts (left panel) pulled down endogenous MKS1 from ARPE19 whole cell extract. (e) Co-immunoprecipitation (co-IP) of endogenous UBE2E1 by rabbit polyclonal anti-MKS1, but not pre-immune serum or an irrelevant antibody (Ab; anti cmyc); IgG light chain (LC) is indicated. (f) Co-IP of exogenously expressed cmyc-MKS1 by anti- UBE2E1 but not pre-immune serum or an irrelevant antibody.

Journal: eLife

Article Title: Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

doi: 10.7554/elife.57593

Figure Lengend Snippet: Figure 2. The E2 ubiquitin conjugation enzyme UBE2E1 interacts with MKS1. (a) Domain structure of MKS1 and UBE2E1 proteins for the indicated isoform showing the locations of the B9/C2 domain, putative ubiquitinated lysines in blue (predicted by UbPred), a predicted coiled-coil (CC) motif, and the E2 ubiquitin (UBQ) conjugation domain in UBE2E1. Numbering indicates the amino acid residue. Dashed lines indicate the region used as ‘bait’ in MKS1 for the yeast two-hybrid assay and the ‘prey’ clones in the UBE2E1 interactant. (b) List of preys identified in the MKS1 Y2H screen (c) Left panel: yeast ‘one-to-one’ assays for the indicated bait, prey and control constructs. Right panel: only colonies for the positive control (p53+ SV40 large T) and MKS1 bait+ UBE2E1 prey grew on triple dropout (-Leu -Trp -His) medium. (d) GST-UBE2E1 purified from bacterial extracts (left panel) pulled down endogenous MKS1 from ARPE19 whole cell extract. (e) Co-immunoprecipitation (co-IP) of endogenous UBE2E1 by rabbit polyclonal anti-MKS1, but not pre-immune serum or an irrelevant antibody (Ab; anti cmyc); IgG light chain (LC) is indicated. (f) Co-IP of exogenously expressed cmyc-MKS1 by anti- UBE2E1 but not pre-immune serum or an irrelevant antibody.

Article Snippet: DOI: https://doi.org/10.7554/eLife.57593 16 of 24 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- mono- and polyubiquitinylated conjugates, clone FK2 (mouse monoclonal) Enzo Life Sciences, Inc ENZ- ABS840 RRID:AB_10541840 WB: 1:1,000 Antibody Anti- 20S proteasome α7 subunit, clone MCP72 (rabbit monoclonal) Enzo Life Sciences Inc BML- PW8110 RRID:AB_10538395 WB: 1:1,000 IF: 1:100 Antibody Anti- MKS1 (rabbit polyclonal) Dawe et al., 2007; Näthke et al., 1996 WB: 1:500 IF: 1:100 Antibody Anti MKS1 (rabbit polyclonal) Proteintech 16206–1- AP RRID:AB_10637856 WB: 1:500 IF: 1:100 Antibody Anti- ubiquitin, clone P4D1 (mouse monoclonal) Santa Cruz Biotechnology, Inc sc- 8017 RRID:AB_2762364 Peptide, recombinant protein MKS1- HIS Proteintech Group, Inc Ag9504 Peptide, recombinant protein UBE2E1- HIS Enzo Life Sciences, Inc UW8710 Peptide, recombinant protein RNF34- HIS Novus Biologicals NBP2- 23440 Peptide, recombinant protein β-catenin- GST Abcam Ab63175 Commercial assay or kit 20 S fluorophore substrate Suc- LLVYAMC Enzo Life Sciences Inc BML- P802- 0005 Commercial assay or kit Dual- Luciferase Reporter Assay system Promega Corp. E1910 Commercial assay or kit Ubiquitination kit Enzo Life Sciences, Inc BML- UW0400 Commercial assay or kit TUBE assays LifeSensors, Malvern, PA, USA UM- 402 Chemical compound, drug MG- 132 Sigma- Aldrich Co. Ltd. C2211 treatment at 10 μM for 3 hr Software, algorithm Prism7 GraphPad Software Inc Continued Informed consent for use of patients in research Informed consent was obtained from all participating families or patients, with studies approved by the Leeds (East) Research Ethics Committee (REC no. 08 /H1306/85) on 4th July 2008.

Techniques: Ubiquitin Proteomics, Conjugation Assay, Residue, Y2H Assay, Clone Assay, Control, Construct, Positive Control, Purification, Immunoprecipitation, Co-Immunoprecipitation Assay

Figure 6. MKS1 is ubiquitinated and its ubiquitynation depends on UBE2E1. (a) shScr and shUbe2e1 mIMCD3 knockdown cells transiently transfected with cmyc-MKS1 and/or FLAG-cmyc-UBE2E1 under different conditions of ciliogenesis: proliferating cells grown in media containing serum (+); ciliated cells grown in the absence of serum (-); and cells undergoing ciliary resorption grown in the absence of serum followed by 2 hr incubation in media with serum (-/+). Increased levels of cmyc-MKS1 and smears representing poly-ubiquitinated (polyUb) cmyc-MKS1 in shUbe2e1 cells are indicated. Addition of exogenous FLAG-cmyc-UBE2E1 partially rescued correct MKS1 levels and ubiquitination. Normalised band intensities for the whole cmyc-MKS1 staining and only polyUb-cmyc-MKS1 are shown below the blots. Bar graph quantitates cmyc-MKS1 levels normalised to β-actin levels for three independent biological replicates. Data was analysed by two-way ANOVA followed by Tukey’s multiple comparison test (statistical significance of comparison between shScr and shUbe2e1 is p < 0.05, error bars represent s.d.). (b) TUBE experiment confirming ubiquitination of cmyc-MKS1. Consistently increased levels of polyubiquitinated cmyc-MKS1 were observed in shUbe2e1 knockdown cells. Broad-range deubiquitinating enzymes (+ DUB) and K63-specific (+ K63 DUB) deubiquitinating enzyme were used to assess the type of MKS1 ubiquitination. Normalised band intensities are shown below the blots. (c) In vitro ubiquitination assay for MKS1-HIS, UBE2E1-HIS, RNF34-HIS, E1-HIS, Ub, and β-catenin-GST fusion proteins. The MKS1 blot shows possible mono-ubiquitination of MKS1 (red asterisk) in the presence of UBE2E1 and RNF34. Auto-ubiquitination of UBE2E1 (green asterisks indicate the addition of one, two, three, four and poly-ubiquitin chains) was inhibited by MKS1. This was further inhibited by addition of β-catenin, but β-catenin addition by itself did not affect UBE2E1 polyubiquitination. although β-catenin was polyubiquitinated by UBE2E1.

Journal: eLife

Article Title: Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

doi: 10.7554/elife.57593

Figure Lengend Snippet: Figure 6. MKS1 is ubiquitinated and its ubiquitynation depends on UBE2E1. (a) shScr and shUbe2e1 mIMCD3 knockdown cells transiently transfected with cmyc-MKS1 and/or FLAG-cmyc-UBE2E1 under different conditions of ciliogenesis: proliferating cells grown in media containing serum (+); ciliated cells grown in the absence of serum (-); and cells undergoing ciliary resorption grown in the absence of serum followed by 2 hr incubation in media with serum (-/+). Increased levels of cmyc-MKS1 and smears representing poly-ubiquitinated (polyUb) cmyc-MKS1 in shUbe2e1 cells are indicated. Addition of exogenous FLAG-cmyc-UBE2E1 partially rescued correct MKS1 levels and ubiquitination. Normalised band intensities for the whole cmyc-MKS1 staining and only polyUb-cmyc-MKS1 are shown below the blots. Bar graph quantitates cmyc-MKS1 levels normalised to β-actin levels for three independent biological replicates. Data was analysed by two-way ANOVA followed by Tukey’s multiple comparison test (statistical significance of comparison between shScr and shUbe2e1 is p < 0.05, error bars represent s.d.). (b) TUBE experiment confirming ubiquitination of cmyc-MKS1. Consistently increased levels of polyubiquitinated cmyc-MKS1 were observed in shUbe2e1 knockdown cells. Broad-range deubiquitinating enzymes (+ DUB) and K63-specific (+ K63 DUB) deubiquitinating enzyme were used to assess the type of MKS1 ubiquitination. Normalised band intensities are shown below the blots. (c) In vitro ubiquitination assay for MKS1-HIS, UBE2E1-HIS, RNF34-HIS, E1-HIS, Ub, and β-catenin-GST fusion proteins. The MKS1 blot shows possible mono-ubiquitination of MKS1 (red asterisk) in the presence of UBE2E1 and RNF34. Auto-ubiquitination of UBE2E1 (green asterisks indicate the addition of one, two, three, four and poly-ubiquitin chains) was inhibited by MKS1. This was further inhibited by addition of β-catenin, but β-catenin addition by itself did not affect UBE2E1 polyubiquitination. although β-catenin was polyubiquitinated by UBE2E1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.57593 16 of 24 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- mono- and polyubiquitinylated conjugates, clone FK2 (mouse monoclonal) Enzo Life Sciences, Inc ENZ- ABS840 RRID:AB_10541840 WB: 1:1,000 Antibody Anti- 20S proteasome α7 subunit, clone MCP72 (rabbit monoclonal) Enzo Life Sciences Inc BML- PW8110 RRID:AB_10538395 WB: 1:1,000 IF: 1:100 Antibody Anti- MKS1 (rabbit polyclonal) Dawe et al., 2007; Näthke et al., 1996 WB: 1:500 IF: 1:100 Antibody Anti MKS1 (rabbit polyclonal) Proteintech 16206–1- AP RRID:AB_10637856 WB: 1:500 IF: 1:100 Antibody Anti- ubiquitin, clone P4D1 (mouse monoclonal) Santa Cruz Biotechnology, Inc sc- 8017 RRID:AB_2762364 Peptide, recombinant protein MKS1- HIS Proteintech Group, Inc Ag9504 Peptide, recombinant protein UBE2E1- HIS Enzo Life Sciences, Inc UW8710 Peptide, recombinant protein RNF34- HIS Novus Biologicals NBP2- 23440 Peptide, recombinant protein β-catenin- GST Abcam Ab63175 Commercial assay or kit 20 S fluorophore substrate Suc- LLVYAMC Enzo Life Sciences Inc BML- P802- 0005 Commercial assay or kit Dual- Luciferase Reporter Assay system Promega Corp. E1910 Commercial assay or kit Ubiquitination kit Enzo Life Sciences, Inc BML- UW0400 Commercial assay or kit TUBE assays LifeSensors, Malvern, PA, USA UM- 402 Chemical compound, drug MG- 132 Sigma- Aldrich Co. Ltd. C2211 treatment at 10 μM for 3 hr Software, algorithm Prism7 GraphPad Software Inc Continued Informed consent for use of patients in research Informed consent was obtained from all participating families or patients, with studies approved by the Leeds (East) Research Ethics Committee (REC no. 08 /H1306/85) on 4th July 2008.

Techniques: Knockdown, Transfection, Incubation, Ubiquitin Proteomics, Staining, Comparison, In Vitro