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  • 93
    R&D Systems mg132
    SENP1 deSUMOylates IDOL and reduces IDOL abundance. A , HEK293T cells were transfected with plasmids expressing Flag-tagged SENP1 or USP19. After 48 h, cells were harvested and IPed with anti-FLAG M2 agarose beads. Bound proteins were eluted by 3×FLAG peptides and analyzed by immunoblotting. B – C , equal amounts of SENP1 or USP19 in TBS (vehicle control) were incubated with 1 μM SUMO1-AMC ( B ) or 0.5 μM ubiquitin (Ub)-AFC ( C ), and released fluorescence of free AMC or AFC was analyzed. D , Huh7 cells were transfected as indicated. After 48 h, cells were treated with 10 μM <t>MG132</t> for 3 h and harvested for the SUMOylation assay as described in Figure 1 . Asterisk indicates nonspecific bands. E , Huh7 cells were transfected as indicated. After 48 h, cells were harvested for immunoblotting analysis. F , densitometric analysis of the IDOL bands in ( E ). Values are normalized to the value obtained from cells transfected with IDOL and UBC9 plus SUMO1 and presented as mean ± SEM (n = 3 independent experiments). ∗∗ p
    Mg132, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Alomone Labs ica 27243 n 6 chloro pyridin 3 yl 3 4 difluoro benzamide
    SENP1 deSUMOylates IDOL and reduces IDOL abundance. A , HEK293T cells were transfected with plasmids expressing Flag-tagged SENP1 or USP19. After 48 h, cells were harvested and IPed with anti-FLAG M2 agarose beads. Bound proteins were eluted by 3×FLAG peptides and analyzed by immunoblotting. B – C , equal amounts of SENP1 or USP19 in TBS (vehicle control) were incubated with 1 μM SUMO1-AMC ( B ) or 0.5 μM ubiquitin (Ub)-AFC ( C ), and released fluorescence of free AMC or AFC was analyzed. D , Huh7 cells were transfected as indicated. After 48 h, cells were treated with 10 μM <t>MG132</t> for 3 h and harvested for the SUMOylation assay as described in Figure 1 . Asterisk indicates nonspecific bands. E , Huh7 cells were transfected as indicated. After 48 h, cells were harvested for immunoblotting analysis. F , densitometric analysis of the IDOL bands in ( E ). Values are normalized to the value obtained from cells transfected with IDOL and UBC9 plus SUMO1 and presented as mean ± SEM (n = 3 independent experiments). ∗∗ p
    Ica 27243 N 6 Chloro Pyridin 3 Yl 3 4 Difluoro Benzamide, supplied by Alomone Labs, 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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    99
    Thermo Fisher sybr green i
    SENP1 deSUMOylates IDOL and reduces IDOL abundance. A , HEK293T cells were transfected with plasmids expressing Flag-tagged SENP1 or USP19. After 48 h, cells were harvested and IPed with anti-FLAG M2 agarose beads. Bound proteins were eluted by 3×FLAG peptides and analyzed by immunoblotting. B – C , equal amounts of SENP1 or USP19 in TBS (vehicle control) were incubated with 1 μM SUMO1-AMC ( B ) or 0.5 μM ubiquitin (Ub)-AFC ( C ), and released fluorescence of free AMC or AFC was analyzed. D , Huh7 cells were transfected as indicated. After 48 h, cells were treated with 10 μM <t>MG132</t> for 3 h and harvested for the SUMOylation assay as described in Figure 1 . Asterisk indicates nonspecific bands. E , Huh7 cells were transfected as indicated. After 48 h, cells were harvested for immunoblotting analysis. F , densitometric analysis of the IDOL bands in ( E ). Values are normalized to the value obtained from cells transfected with IDOL and UBC9 plus SUMO1 and presented as mean ± SEM (n = 3 independent experiments). ∗∗ p
    Sybr Green I, supplied by Thermo Fisher, 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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    97
    Worthington Biochemical collagenase type i
    SENP1 deSUMOylates IDOL and reduces IDOL abundance. A , HEK293T cells were transfected with plasmids expressing Flag-tagged SENP1 or USP19. After 48 h, cells were harvested and IPed with anti-FLAG M2 agarose beads. Bound proteins were eluted by 3×FLAG peptides and analyzed by immunoblotting. B – C , equal amounts of SENP1 or USP19 in TBS (vehicle control) were incubated with 1 μM SUMO1-AMC ( B ) or 0.5 μM ubiquitin (Ub)-AFC ( C ), and released fluorescence of free AMC or AFC was analyzed. D , Huh7 cells were transfected as indicated. After 48 h, cells were treated with 10 μM <t>MG132</t> for 3 h and harvested for the SUMOylation assay as described in Figure 1 . Asterisk indicates nonspecific bands. E , Huh7 cells were transfected as indicated. After 48 h, cells were harvested for immunoblotting analysis. F , densitometric analysis of the IDOL bands in ( E ). Values are normalized to the value obtained from cells transfected with IDOL and UBC9 plus SUMO1 and presented as mean ± SEM (n = 3 independent experiments). ∗∗ p
    Collagenase Type I, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    SENP1 deSUMOylates IDOL and reduces IDOL abundance. A , HEK293T cells were transfected with plasmids expressing Flag-tagged SENP1 or USP19. After 48 h, cells were harvested and IPed with anti-FLAG M2 agarose beads. Bound proteins were eluted by 3×FLAG peptides and analyzed by immunoblotting. B – C , equal amounts of SENP1 or USP19 in TBS (vehicle control) were incubated with 1 μM SUMO1-AMC ( B ) or 0.5 μM ubiquitin (Ub)-AFC ( C ), and released fluorescence of free AMC or AFC was analyzed. D , Huh7 cells were transfected as indicated. After 48 h, cells were treated with 10 μM MG132 for 3 h and harvested for the SUMOylation assay as described in Figure 1 . Asterisk indicates nonspecific bands. E , Huh7 cells were transfected as indicated. After 48 h, cells were harvested for immunoblotting analysis. F , densitometric analysis of the IDOL bands in ( E ). Values are normalized to the value obtained from cells transfected with IDOL and UBC9 plus SUMO1 and presented as mean ± SEM (n = 3 independent experiments). ∗∗ p

    Journal: The Journal of Biological Chemistry

    Article Title: SUMOylation of the ubiquitin ligase IDOL decreases LDL receptor levels and is reversed by SENP1

    doi: 10.1074/jbc.RA120.015420

    Figure Lengend Snippet: SENP1 deSUMOylates IDOL and reduces IDOL abundance. A , HEK293T cells were transfected with plasmids expressing Flag-tagged SENP1 or USP19. After 48 h, cells were harvested and IPed with anti-FLAG M2 agarose beads. Bound proteins were eluted by 3×FLAG peptides and analyzed by immunoblotting. B – C , equal amounts of SENP1 or USP19 in TBS (vehicle control) were incubated with 1 μM SUMO1-AMC ( B ) or 0.5 μM ubiquitin (Ub)-AFC ( C ), and released fluorescence of free AMC or AFC was analyzed. D , Huh7 cells were transfected as indicated. After 48 h, cells were treated with 10 μM MG132 for 3 h and harvested for the SUMOylation assay as described in Figure 1 . Asterisk indicates nonspecific bands. E , Huh7 cells were transfected as indicated. After 48 h, cells were harvested for immunoblotting analysis. F , densitometric analysis of the IDOL bands in ( E ). Values are normalized to the value obtained from cells transfected with IDOL and UBC9 plus SUMO1 and presented as mean ± SEM (n = 3 independent experiments). ∗∗ p

    Article Snippet: SUMO1-AMC (#UL-704), ubiquitin-AFC (#U-551-050), and MG132 (#I-130) were from Boston Biochem.

    Techniques: Transfection, Expressing, Incubation, Fluorescence

    IDOL is conjugated with SUMO1 primarily at the K293 residue. A , alignment of the IDOL sequence across species with two inverted SUMOylation consensus motifs E/DxKψ (where x stands for any amino acid and ψ for a hydrophobic amino acid) shaded in gray. The K20 and K293 residues are in red. B – D , Huh7 cells were transfected with the indicated plasmids. After 48 h, cells were treated with 10 μM MG132 for 3 h and harvested. Lysates were pulled down by Ni-NTA agarose and then subjected to immunoblotting with the indicated antibodies. Asterisks indicate nonspecific bands. E , densitometric analysis of the high-molecular-weight smears relative to the unmodified IDOL band as shown in ( D ). Values are normalized to the value obtained from cells transfected with the wild-type form of IDOL only and presented as mean ± SEM (n = 3 independent experiments). ∗∗ p

    Journal: The Journal of Biological Chemistry

    Article Title: SUMOylation of the ubiquitin ligase IDOL decreases LDL receptor levels and is reversed by SENP1

    doi: 10.1074/jbc.RA120.015420

    Figure Lengend Snippet: IDOL is conjugated with SUMO1 primarily at the K293 residue. A , alignment of the IDOL sequence across species with two inverted SUMOylation consensus motifs E/DxKψ (where x stands for any amino acid and ψ for a hydrophobic amino acid) shaded in gray. The K20 and K293 residues are in red. B – D , Huh7 cells were transfected with the indicated plasmids. After 48 h, cells were treated with 10 μM MG132 for 3 h and harvested. Lysates were pulled down by Ni-NTA agarose and then subjected to immunoblotting with the indicated antibodies. Asterisks indicate nonspecific bands. E , densitometric analysis of the high-molecular-weight smears relative to the unmodified IDOL band as shown in ( D ). Values are normalized to the value obtained from cells transfected with the wild-type form of IDOL only and presented as mean ± SEM (n = 3 independent experiments). ∗∗ p

    Article Snippet: SUMO1-AMC (#UL-704), ubiquitin-AFC (#U-551-050), and MG132 (#I-130) were from Boston Biochem.

    Techniques: Sequencing, Transfection, Molecular Weight