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  • 89
    Santa Cruz Biotechnology ub p4d1
    Analysis of EGFR ubiquitination. ( A ) HeLa or NR6 cells were stimulated with the indicated concentrations of EGF for 2 min (in this and all subsequent figures). IP and IB were performed as indicated (Ub, ubiquitin <t>P4D1</t> antibody). ( B ) HeLa cells were stimulated with EGF, followed by IB with the indicated antibodies. ( C , D ) Lysates of HeLa cells stimulated with EGF, as indicated, were subjected to ELISA, forward approach ( Supplementary Figure 2A ), using the indicated detecting Ab (Ub, FK2 antibody). Results are shown as % of max (see Materials and methods). ( E ) Lysates of HeLa cells stimulated with EGF, as indicated, were subjected to ELISA, reverse approach ( Supplementary Figure 2B ), using the indicated detecting Ab (Ub, FK2 antibody). ( F ) Comparison of the EGFR ubiquitination and phosphorylation curves of HeLa cells obtained by forward and reverse ELISA. In all panels (and in all subsequent figures), error bars indicate s.d. calculated on at least three independent experiments. P -values were calculated using two-way ANOVA analysis. When comparing curves that showed significant differences (in all figures), we show the relative P -values; when comparing curves that did not show significant differences (in all figures), we display R , the Pearson correlation coefficient. Source data for this figure is available on the online supplementary information page.
    Ub P4d1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 211 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology mouse anti ub p4d1
    FcεRI is preferentially monoubiquitinated at multiple sites upon Ag binding. (A) The ability of <t>P4D1</t> and FK1 anti-Ub mAbs to recognize a single Ub molecule or a poly-Ub chain, respectively, was tested by Western blotting using an Ub ladder. (B) RBL-2H3 cells (4×10 7 /sample) were sensitized with anti-DNP IgE, and stimulated (+) or not (−) with DNP-HSA (Ag) for 1 min at 37°C. Total cell lysates (TCL) and anti-IgE immunoprecipitates were resolved by SDS-PAGE and immunoblotted with the indicated Abs.
    Mouse Anti Ub P4d1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc anti ub p4d1
    FcεRI is preferentially monoubiquitinated at multiple sites upon Ag binding. (A) The ability of <t>P4D1</t> and FK1 anti-Ub mAbs to recognize a single Ub molecule or a poly-Ub chain, respectively, was tested by Western blotting using an Ub ladder. (B) RBL-2H3 cells (4×10 7 /sample) were sensitized with anti-DNP IgE, and stimulated (+) or not (−) with DNP-HSA (Ag) for 1 min at 37°C. Total cell lysates (TCL) and anti-IgE immunoprecipitates were resolved by SDS-PAGE and immunoblotted with the indicated Abs.
    Anti Ub P4d1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc anti ub p4d1 antibody
    CD133 undergoes monoubiquitination. (A, E, and G) U87MG cells were infected with a lentivirus expressing Flag (control) or CD133-Flag. (A) CD133 expression was analyzed by Western blotting; β-actin was blotted as a loading control. IP analysis using Flag-M2 was performed to determine the ubiquitination of CD133. Ubiquitin protein was added to neutralizing the primary ubiquitin antibody (bottom panel). (B) HEK293T cells were transfected with a Flag (control) or CD133-Flag plasmid, and IP analysis was performed to determine the ubiquitination of CD133. β-Actin was blotted as a loading control. (C) A differentiation assay was performed. GBM1 cells were cultured in 2% FBS medium for 7 days, and the characteristics of CSCs were evaluated. Representative images of GBM1 cells during the differentiation assay are shown. Bars = 150 μm. (D) Endogenous CD133 from GBM1 sphere culture cells was precipitated by use of CD133 antibody. Normal mouse IgG antibody was used as a negative control. Ubiquitination of CD133 protein was detected by Western blotting; β-actin was blotted as an internal control. (E) CD133 from U87MG cells expressing CD133-Flag was precipitated by use of a Flag antibody. <t>Ub-P4D1</t> antibody and Ub-FK1 antibody were used to detecting the type of CD133 ubiquitination. (F) HEK293T cells coexpressing CD133-Flag and either the HA-Ub-WT or HA-Ub-KO plasmid. CD133 ubiquitination was detected by Western blotting following co-IP assays. (G) U87MG cells expressing CD133-Flag were subjected to IP with an anti-Flag antibody, and CD133 protein was treated with PNGase F for deglycosylation and then immunoblotted with anti-CD133 or anti-Ub (P4D1) antibodies. Whole-cell lysates were analyzed by immunoblotting, with CD133 and β-actin as input. All results were collected from three independent experiments. N.Ms IgG, normal mouse IgG; IP, immunoprecipitation; Ub-WT, wild-type ubiquitin; Ub-KO, all 7 ubiquitin lysines were mutated to arginine; Exp., exposure.
    Anti Ub P4d1 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Covance anti ub p4d1
    CD133 undergoes monoubiquitination. (A, E, and G) U87MG cells were infected with a lentivirus expressing Flag (control) or CD133-Flag. (A) CD133 expression was analyzed by Western blotting; β-actin was blotted as a loading control. IP analysis using Flag-M2 was performed to determine the ubiquitination of CD133. Ubiquitin protein was added to neutralizing the primary ubiquitin antibody (bottom panel). (B) HEK293T cells were transfected with a Flag (control) or CD133-Flag plasmid, and IP analysis was performed to determine the ubiquitination of CD133. β-Actin was blotted as a loading control. (C) A differentiation assay was performed. GBM1 cells were cultured in 2% FBS medium for 7 days, and the characteristics of CSCs were evaluated. Representative images of GBM1 cells during the differentiation assay are shown. Bars = 150 μm. (D) Endogenous CD133 from GBM1 sphere culture cells was precipitated by use of CD133 antibody. Normal mouse IgG antibody was used as a negative control. Ubiquitination of CD133 protein was detected by Western blotting; β-actin was blotted as an internal control. (E) CD133 from U87MG cells expressing CD133-Flag was precipitated by use of a Flag antibody. <t>Ub-P4D1</t> antibody and Ub-FK1 antibody were used to detecting the type of CD133 ubiquitination. (F) HEK293T cells coexpressing CD133-Flag and either the HA-Ub-WT or HA-Ub-KO plasmid. CD133 ubiquitination was detected by Western blotting following co-IP assays. (G) U87MG cells expressing CD133-Flag were subjected to IP with an anti-Flag antibody, and CD133 protein was treated with PNGase F for deglycosylation and then immunoblotted with anti-CD133 or anti-Ub (P4D1) antibodies. Whole-cell lysates were analyzed by immunoblotting, with CD133 and β-actin as input. All results were collected from three independent experiments. N.Ms IgG, normal mouse IgG; IP, immunoprecipitation; Ub-WT, wild-type ubiquitin; Ub-KO, all 7 ubiquitin lysines were mutated to arginine; Exp., exposure.
    Anti Ub P4d1, supplied by Covance, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology ubiquitin ub p4d1 mouse monoclonal antibodies
    CD133 undergoes monoubiquitination. (A, E, and G) U87MG cells were infected with a lentivirus expressing Flag (control) or CD133-Flag. (A) CD133 expression was analyzed by Western blotting; β-actin was blotted as a loading control. IP analysis using Flag-M2 was performed to determine the ubiquitination of CD133. Ubiquitin protein was added to neutralizing the primary ubiquitin antibody (bottom panel). (B) HEK293T cells were transfected with a Flag (control) or CD133-Flag plasmid, and IP analysis was performed to determine the ubiquitination of CD133. β-Actin was blotted as a loading control. (C) A differentiation assay was performed. GBM1 cells were cultured in 2% FBS medium for 7 days, and the characteristics of CSCs were evaluated. Representative images of GBM1 cells during the differentiation assay are shown. Bars = 150 μm. (D) Endogenous CD133 from GBM1 sphere culture cells was precipitated by use of CD133 antibody. Normal mouse IgG antibody was used as a negative control. Ubiquitination of CD133 protein was detected by Western blotting; β-actin was blotted as an internal control. (E) CD133 from U87MG cells expressing CD133-Flag was precipitated by use of a Flag antibody. <t>Ub-P4D1</t> antibody and Ub-FK1 antibody were used to detecting the type of CD133 ubiquitination. (F) HEK293T cells coexpressing CD133-Flag and either the HA-Ub-WT or HA-Ub-KO plasmid. CD133 ubiquitination was detected by Western blotting following co-IP assays. (G) U87MG cells expressing CD133-Flag were subjected to IP with an anti-Flag antibody, and CD133 protein was treated with PNGase F for deglycosylation and then immunoblotted with anti-CD133 or anti-Ub (P4D1) antibodies. Whole-cell lysates were analyzed by immunoblotting, with CD133 and β-actin as input. All results were collected from three independent experiments. N.Ms IgG, normal mouse IgG; IP, immunoprecipitation; Ub-WT, wild-type ubiquitin; Ub-KO, all 7 ubiquitin lysines were mutated to arginine; Exp., exposure.
    Ubiquitin Ub P4d1 Mouse Monoclonal Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology ubiquitin ub p4d1
    CD133 undergoes monoubiquitination. (A, E, and G) U87MG cells were infected with a lentivirus expressing Flag (control) or CD133-Flag. (A) CD133 expression was analyzed by Western blotting; β-actin was blotted as a loading control. IP analysis using Flag-M2 was performed to determine the ubiquitination of CD133. Ubiquitin protein was added to neutralizing the primary ubiquitin antibody (bottom panel). (B) HEK293T cells were transfected with a Flag (control) or CD133-Flag plasmid, and IP analysis was performed to determine the ubiquitination of CD133. β-Actin was blotted as a loading control. (C) A differentiation assay was performed. GBM1 cells were cultured in 2% FBS medium for 7 days, and the characteristics of CSCs were evaluated. Representative images of GBM1 cells during the differentiation assay are shown. Bars = 150 μm. (D) Endogenous CD133 from GBM1 sphere culture cells was precipitated by use of CD133 antibody. Normal mouse IgG antibody was used as a negative control. Ubiquitination of CD133 protein was detected by Western blotting; β-actin was blotted as an internal control. (E) CD133 from U87MG cells expressing CD133-Flag was precipitated by use of a Flag antibody. <t>Ub-P4D1</t> antibody and Ub-FK1 antibody were used to detecting the type of CD133 ubiquitination. (F) HEK293T cells coexpressing CD133-Flag and either the HA-Ub-WT or HA-Ub-KO plasmid. CD133 ubiquitination was detected by Western blotting following co-IP assays. (G) U87MG cells expressing CD133-Flag were subjected to IP with an anti-Flag antibody, and CD133 protein was treated with PNGase F for deglycosylation and then immunoblotted with anti-CD133 or anti-Ub (P4D1) antibodies. Whole-cell lysates were analyzed by immunoblotting, with CD133 and β-actin as input. All results were collected from three independent experiments. N.Ms IgG, normal mouse IgG; IP, immunoprecipitation; Ub-WT, wild-type ubiquitin; Ub-KO, all 7 ubiquitin lysines were mutated to arginine; Exp., exposure.
    Ubiquitin Ub P4d1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Santa Cruz Biotechnology mouse α ub p4d1 mab
    CD133 undergoes monoubiquitination. (A, E, and G) U87MG cells were infected with a lentivirus expressing Flag (control) or CD133-Flag. (A) CD133 expression was analyzed by Western blotting; β-actin was blotted as a loading control. IP analysis using Flag-M2 was performed to determine the ubiquitination of CD133. Ubiquitin protein was added to neutralizing the primary ubiquitin antibody (bottom panel). (B) HEK293T cells were transfected with a Flag (control) or CD133-Flag plasmid, and IP analysis was performed to determine the ubiquitination of CD133. β-Actin was blotted as a loading control. (C) A differentiation assay was performed. GBM1 cells were cultured in 2% FBS medium for 7 days, and the characteristics of CSCs were evaluated. Representative images of GBM1 cells during the differentiation assay are shown. Bars = 150 μm. (D) Endogenous CD133 from GBM1 sphere culture cells was precipitated by use of CD133 antibody. Normal mouse IgG antibody was used as a negative control. Ubiquitination of CD133 protein was detected by Western blotting; β-actin was blotted as an internal control. (E) CD133 from U87MG cells expressing CD133-Flag was precipitated by use of a Flag antibody. <t>Ub-P4D1</t> antibody and Ub-FK1 antibody were used to detecting the type of CD133 ubiquitination. (F) HEK293T cells coexpressing CD133-Flag and either the HA-Ub-WT or HA-Ub-KO plasmid. CD133 ubiquitination was detected by Western blotting following co-IP assays. (G) U87MG cells expressing CD133-Flag were subjected to IP with an anti-Flag antibody, and CD133 protein was treated with PNGase F for deglycosylation and then immunoblotted with anti-CD133 or anti-Ub (P4D1) antibodies. Whole-cell lysates were analyzed by immunoblotting, with CD133 and β-actin as input. All results were collected from three independent experiments. N.Ms IgG, normal mouse IgG; IP, immunoprecipitation; Ub-WT, wild-type ubiquitin; Ub-KO, all 7 ubiquitin lysines were mutated to arginine; Exp., exposure.
    Mouse α Ub P4d1 Mab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Santa Cruz Biotechnology ub p4d1 agrose slurry
    CD133 undergoes monoubiquitination. (A, E, and G) U87MG cells were infected with a lentivirus expressing Flag (control) or CD133-Flag. (A) CD133 expression was analyzed by Western blotting; β-actin was blotted as a loading control. IP analysis using Flag-M2 was performed to determine the ubiquitination of CD133. Ubiquitin protein was added to neutralizing the primary ubiquitin antibody (bottom panel). (B) HEK293T cells were transfected with a Flag (control) or CD133-Flag plasmid, and IP analysis was performed to determine the ubiquitination of CD133. β-Actin was blotted as a loading control. (C) A differentiation assay was performed. GBM1 cells were cultured in 2% FBS medium for 7 days, and the characteristics of CSCs were evaluated. Representative images of GBM1 cells during the differentiation assay are shown. Bars = 150 μm. (D) Endogenous CD133 from GBM1 sphere culture cells was precipitated by use of CD133 antibody. Normal mouse IgG antibody was used as a negative control. Ubiquitination of CD133 protein was detected by Western blotting; β-actin was blotted as an internal control. (E) CD133 from U87MG cells expressing CD133-Flag was precipitated by use of a Flag antibody. <t>Ub-P4D1</t> antibody and Ub-FK1 antibody were used to detecting the type of CD133 ubiquitination. (F) HEK293T cells coexpressing CD133-Flag and either the HA-Ub-WT or HA-Ub-KO plasmid. CD133 ubiquitination was detected by Western blotting following co-IP assays. (G) U87MG cells expressing CD133-Flag were subjected to IP with an anti-Flag antibody, and CD133 protein was treated with PNGase F for deglycosylation and then immunoblotted with anti-CD133 or anti-Ub (P4D1) antibodies. Whole-cell lysates were analyzed by immunoblotting, with CD133 and β-actin as input. All results were collected from three independent experiments. N.Ms IgG, normal mouse IgG; IP, immunoprecipitation; Ub-WT, wild-type ubiquitin; Ub-KO, all 7 ubiquitin lysines were mutated to arginine; Exp., exposure.
    Ub P4d1 Agrose Slurry, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology ub p4d1 mouse
    CD133 undergoes monoubiquitination. (A, E, and G) U87MG cells were infected with a lentivirus expressing Flag (control) or CD133-Flag. (A) CD133 expression was analyzed by Western blotting; β-actin was blotted as a loading control. IP analysis using Flag-M2 was performed to determine the ubiquitination of CD133. Ubiquitin protein was added to neutralizing the primary ubiquitin antibody (bottom panel). (B) HEK293T cells were transfected with a Flag (control) or CD133-Flag plasmid, and IP analysis was performed to determine the ubiquitination of CD133. β-Actin was blotted as a loading control. (C) A differentiation assay was performed. GBM1 cells were cultured in 2% FBS medium for 7 days, and the characteristics of CSCs were evaluated. Representative images of GBM1 cells during the differentiation assay are shown. Bars = 150 μm. (D) Endogenous CD133 from GBM1 sphere culture cells was precipitated by use of CD133 antibody. Normal mouse IgG antibody was used as a negative control. Ubiquitination of CD133 protein was detected by Western blotting; β-actin was blotted as an internal control. (E) CD133 from U87MG cells expressing CD133-Flag was precipitated by use of a Flag antibody. <t>Ub-P4D1</t> antibody and Ub-FK1 antibody were used to detecting the type of CD133 ubiquitination. (F) HEK293T cells coexpressing CD133-Flag and either the HA-Ub-WT or HA-Ub-KO plasmid. CD133 ubiquitination was detected by Western blotting following co-IP assays. (G) U87MG cells expressing CD133-Flag were subjected to IP with an anti-Flag antibody, and CD133 protein was treated with PNGase F for deglycosylation and then immunoblotted with anti-CD133 or anti-Ub (P4D1) antibodies. Whole-cell lysates were analyzed by immunoblotting, with CD133 and β-actin as input. All results were collected from three independent experiments. N.Ms IgG, normal mouse IgG; IP, immunoprecipitation; Ub-WT, wild-type ubiquitin; Ub-KO, all 7 ubiquitin lysines were mutated to arginine; Exp., exposure.
    Ub P4d1 Mouse, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Analysis of EGFR ubiquitination. ( A ) HeLa or NR6 cells were stimulated with the indicated concentrations of EGF for 2 min (in this and all subsequent figures). IP and IB were performed as indicated (Ub, ubiquitin P4D1 antibody). ( B ) HeLa cells were stimulated with EGF, followed by IB with the indicated antibodies. ( C , D ) Lysates of HeLa cells stimulated with EGF, as indicated, were subjected to ELISA, forward approach ( Supplementary Figure 2A ), using the indicated detecting Ab (Ub, FK2 antibody). Results are shown as % of max (see Materials and methods). ( E ) Lysates of HeLa cells stimulated with EGF, as indicated, were subjected to ELISA, reverse approach ( Supplementary Figure 2B ), using the indicated detecting Ab (Ub, FK2 antibody). ( F ) Comparison of the EGFR ubiquitination and phosphorylation curves of HeLa cells obtained by forward and reverse ELISA. In all panels (and in all subsequent figures), error bars indicate s.d. calculated on at least three independent experiments. P -values were calculated using two-way ANOVA analysis. When comparing curves that showed significant differences (in all figures), we show the relative P -values; when comparing curves that did not show significant differences (in all figures), we display R , the Pearson correlation coefficient. Source data for this figure is available on the online supplementary information page.

    Journal: The EMBO Journal

    Article Title: Threshold-controlled ubiquitination of the EGFR directs receptor fate

    doi: 10.1038/emboj.2013.149

    Figure Lengend Snippet: Analysis of EGFR ubiquitination. ( A ) HeLa or NR6 cells were stimulated with the indicated concentrations of EGF for 2 min (in this and all subsequent figures). IP and IB were performed as indicated (Ub, ubiquitin P4D1 antibody). ( B ) HeLa cells were stimulated with EGF, followed by IB with the indicated antibodies. ( C , D ) Lysates of HeLa cells stimulated with EGF, as indicated, were subjected to ELISA, forward approach ( Supplementary Figure 2A ), using the indicated detecting Ab (Ub, FK2 antibody). Results are shown as % of max (see Materials and methods). ( E ) Lysates of HeLa cells stimulated with EGF, as indicated, were subjected to ELISA, reverse approach ( Supplementary Figure 2B ), using the indicated detecting Ab (Ub, FK2 antibody). ( F ) Comparison of the EGFR ubiquitination and phosphorylation curves of HeLa cells obtained by forward and reverse ELISA. In all panels (and in all subsequent figures), error bars indicate s.d. calculated on at least three independent experiments. P -values were calculated using two-way ANOVA analysis. When comparing curves that showed significant differences (in all figures), we show the relative P -values; when comparing curves that did not show significant differences (in all figures), we display R , the Pearson correlation coefficient. Source data for this figure is available on the online supplementary information page.

    Article Snippet: Antibodies were: a polyclonal anti-EGFR (made in house, directed against aa 1172–1186 of human EGFR), two monoclonal anti-EGFR antibodies directed against the extracellular domain of human EGFR (m108 hybridoma, American Type Culture Collection (ATCC), ( ) and Ab-1, Calbiochem), anti-EGFR phospho-specific antibodies (Cell Signaling), anti-pY (4G10, Upstate), anti-Ub P4D1 (Santa Cruz, used in all anti-Ub IBs, unless otherwise specified), anti-Ub FK2 (BIOMOL, used in all ELISAs, see , and in some control experiments, as indicated in the figures), anti-Ub ZTA10 (made in house, monoclonal; used in some control experiments, as indicated in the figures), anti-tubulin (Santa Cruz), anti-vinculin (Sigma), anti-dynamin (Santa Cruz), anti-Grb2 (Santa Cruz and BD), anti-c-Cbl (Santa Cruz and BD), anti-Cbl-b (BD), anti-Shc (BD), anti-GST (in-house polyclonal).

    Techniques: Enzyme-linked Immunosorbent Assay, Polyacrylamide Gel Electrophoresis

    FcεRI is preferentially monoubiquitinated at multiple sites upon Ag binding. (A) The ability of P4D1 and FK1 anti-Ub mAbs to recognize a single Ub molecule or a poly-Ub chain, respectively, was tested by Western blotting using an Ub ladder. (B) RBL-2H3 cells (4×10 7 /sample) were sensitized with anti-DNP IgE, and stimulated (+) or not (−) with DNP-HSA (Ag) for 1 min at 37°C. Total cell lysates (TCL) and anti-IgE immunoprecipitates were resolved by SDS-PAGE and immunoblotted with the indicated Abs.

    Journal: PLoS ONE

    Article Title: Lipid Raft-Dependent Fc?RI Ubiquitination Regulates Receptor Endocytosis through the Action of Ubiquitin Binding Adaptors

    doi: 10.1371/journal.pone.0005604

    Figure Lengend Snippet: FcεRI is preferentially monoubiquitinated at multiple sites upon Ag binding. (A) The ability of P4D1 and FK1 anti-Ub mAbs to recognize a single Ub molecule or a poly-Ub chain, respectively, was tested by Western blotting using an Ub ladder. (B) RBL-2H3 cells (4×10 7 /sample) were sensitized with anti-DNP IgE, and stimulated (+) or not (−) with DNP-HSA (Ag) for 1 min at 37°C. Total cell lysates (TCL) and anti-IgE immunoprecipitates were resolved by SDS-PAGE and immunoblotted with the indicated Abs.

    Article Snippet: Rabbit anti-Eps15 and anti-Lyn 44 polyclonal Abs and mouse anti-Ub P4D1 and anti-clathrin heavy chain mAbs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Binding Assay, Western Blot, SDS Page

    CD133 undergoes monoubiquitination. (A, E, and G) U87MG cells were infected with a lentivirus expressing Flag (control) or CD133-Flag. (A) CD133 expression was analyzed by Western blotting; β-actin was blotted as a loading control. IP analysis using Flag-M2 was performed to determine the ubiquitination of CD133. Ubiquitin protein was added to neutralizing the primary ubiquitin antibody (bottom panel). (B) HEK293T cells were transfected with a Flag (control) or CD133-Flag plasmid, and IP analysis was performed to determine the ubiquitination of CD133. β-Actin was blotted as a loading control. (C) A differentiation assay was performed. GBM1 cells were cultured in 2% FBS medium for 7 days, and the characteristics of CSCs were evaluated. Representative images of GBM1 cells during the differentiation assay are shown. Bars = 150 μm. (D) Endogenous CD133 from GBM1 sphere culture cells was precipitated by use of CD133 antibody. Normal mouse IgG antibody was used as a negative control. Ubiquitination of CD133 protein was detected by Western blotting; β-actin was blotted as an internal control. (E) CD133 from U87MG cells expressing CD133-Flag was precipitated by use of a Flag antibody. Ub-P4D1 antibody and Ub-FK1 antibody were used to detecting the type of CD133 ubiquitination. (F) HEK293T cells coexpressing CD133-Flag and either the HA-Ub-WT or HA-Ub-KO plasmid. CD133 ubiquitination was detected by Western blotting following co-IP assays. (G) U87MG cells expressing CD133-Flag were subjected to IP with an anti-Flag antibody, and CD133 protein was treated with PNGase F for deglycosylation and then immunoblotted with anti-CD133 or anti-Ub (P4D1) antibodies. Whole-cell lysates were analyzed by immunoblotting, with CD133 and β-actin as input. All results were collected from three independent experiments. N.Ms IgG, normal mouse IgG; IP, immunoprecipitation; Ub-WT, wild-type ubiquitin; Ub-KO, all 7 ubiquitin lysines were mutated to arginine; Exp., exposure.

    Journal: Molecular and Cellular Biology

    Article Title: Monoubiquitination of Cancer Stem Cell Marker CD133 at Lysine 848 Regulates Its Secretion and Promotes Cell Migration

    doi: 10.1128/MCB.00024-18

    Figure Lengend Snippet: CD133 undergoes monoubiquitination. (A, E, and G) U87MG cells were infected with a lentivirus expressing Flag (control) or CD133-Flag. (A) CD133 expression was analyzed by Western blotting; β-actin was blotted as a loading control. IP analysis using Flag-M2 was performed to determine the ubiquitination of CD133. Ubiquitin protein was added to neutralizing the primary ubiquitin antibody (bottom panel). (B) HEK293T cells were transfected with a Flag (control) or CD133-Flag plasmid, and IP analysis was performed to determine the ubiquitination of CD133. β-Actin was blotted as a loading control. (C) A differentiation assay was performed. GBM1 cells were cultured in 2% FBS medium for 7 days, and the characteristics of CSCs were evaluated. Representative images of GBM1 cells during the differentiation assay are shown. Bars = 150 μm. (D) Endogenous CD133 from GBM1 sphere culture cells was precipitated by use of CD133 antibody. Normal mouse IgG antibody was used as a negative control. Ubiquitination of CD133 protein was detected by Western blotting; β-actin was blotted as an internal control. (E) CD133 from U87MG cells expressing CD133-Flag was precipitated by use of a Flag antibody. Ub-P4D1 antibody and Ub-FK1 antibody were used to detecting the type of CD133 ubiquitination. (F) HEK293T cells coexpressing CD133-Flag and either the HA-Ub-WT or HA-Ub-KO plasmid. CD133 ubiquitination was detected by Western blotting following co-IP assays. (G) U87MG cells expressing CD133-Flag were subjected to IP with an anti-Flag antibody, and CD133 protein was treated with PNGase F for deglycosylation and then immunoblotted with anti-CD133 or anti-Ub (P4D1) antibodies. Whole-cell lysates were analyzed by immunoblotting, with CD133 and β-actin as input. All results were collected from three independent experiments. N.Ms IgG, normal mouse IgG; IP, immunoprecipitation; Ub-WT, wild-type ubiquitin; Ub-KO, all 7 ubiquitin lysines were mutated to arginine; Exp., exposure.

    Article Snippet: Brefeldin A, anti-Ub (P4D1) antibody, anti-LAMP1 antibody, anti-Hrs antibody, anti-STAM antibody, anti-GM130 antibody, anti-HA antibody, and anti-Flag antibody were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Infection, Expressing, Western Blot, Transfection, Plasmid Preparation, Differentiation Assay, Cell Culture, Negative Control, Co-Immunoprecipitation Assay, Mass Spectrometry, Immunoprecipitation