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k48 tetra-ubiquitin (cat# sbb-up0070)  (South Bay Bio)
90
South Bay Bio k48 tetra-ubiquitin (cat# sbb-up0070)
( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, <t>K48,</t> and K63 Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.
K48 Tetra Ubiquitin (Cat# Sbb Up0070), supplied by South Bay Bio, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/k48 tetra-ubiquitin (cat# sbb-up0070)/product/South Bay Bio
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k63 tetra-ubiquitin (cat# sbb-up0073)  (South Bay Bio)
90
South Bay Bio k63 tetra-ubiquitin (cat# sbb-up0073)
( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and <t>K63</t> Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.
K63 Tetra Ubiquitin (Cat# Sbb Up0073), supplied by South Bay Bio, 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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recombinant human k48–linked tetra–ubiquitin  (Boston Biochem)
90
Boston Biochem recombinant human k48–linked tetra–ubiquitin
( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and <t>K63</t> Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.
Recombinant Human K48–Linked Tetra–Ubiquitin, supplied by Boston Biochem, 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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tetra-ubiquitin/ub4 wt chains (k48-linked)  (Boston Biochem)
90
Boston Biochem tetra-ubiquitin/ub4 wt chains (k48-linked)
( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and <t>K63</t> Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.
Tetra Ubiquitin/Ub4 Wt Chains (K48 Linked), supplied by Boston Biochem, 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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k63-linked tetra-ubiquitin chains (ub4  (Adipogen)
90
Adipogen k63-linked tetra-ubiquitin chains (ub4
( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and <t>K63</t> Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.
K63 Linked Tetra Ubiquitin Chains (Ub4, supplied by Adipogen, 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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k63-linked tetra-ubiquitin chains (ub4 - by Bioz Stars, 2026-02
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tetra-k63 ubiquitin  (Boston Biochem)
90
Boston Biochem tetra-k63 ubiquitin
( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and <t>K63</t> Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.
Tetra K63 Ubiquitin, supplied by Boston Biochem, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/tetra-k63 ubiquitin/product/Boston Biochem
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tetra-k63 ubiquitin - by Bioz Stars, 2026-02
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linear k48- and k63-linked tetra-ubiquitin  (Boston Biochem)
90
Boston Biochem linear k48- and k63-linked tetra-ubiquitin
( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and <t>K63</t> Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.
Linear K48 And K63 Linked Tetra Ubiquitin, supplied by Boston Biochem, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/linear k48- and k63-linked tetra-ubiquitin/product/Boston Biochem
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linear k48- and k63-linked tetra-ubiquitin - by Bioz Stars, 2026-02
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tetra ubiquitin rhodamine110  (Boston Biochem)
94
Boston Biochem tetra ubiquitin rhodamine110
( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and <t>K63</t> Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.
Tetra Ubiquitin Rhodamine110, supplied by Boston Biochem, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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tetra ubiquitin rhodamine110 - by Bioz Stars, 2026-02
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tetra‑ubiquitin‑rhodamine110  (LifeSensors)
90
LifeSensors tetra‑ubiquitin‑rhodamine110
( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and <t>K63</t> Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.
Tetra‑Ubiquitin‑Rhodamine110, supplied by LifeSensors, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/tetra‑ubiquitin‑rhodamine110/product/LifeSensors
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tetra‑ubiquitin‑rhodamine110 - by Bioz Stars, 2026-02
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tetra-ubiquitin  (Twist Bioscience)
90
Twist Bioscience tetra-ubiquitin
( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and <t>K63</t> Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.
Tetra Ubiquitin, supplied by Twist Bioscience, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/tetra-ubiquitin/product/Twist Bioscience
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tetra-ubiquitin - by Bioz Stars, 2026-02
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Image Search Results


( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and K63 Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.

Journal: bioRxiv

Article Title: Unanchored ubiquitin chains promote the non-canonical inflammasome via UBXN1

doi: 10.1101/2024.10.30.621131

Figure Lengend Snippet: ( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and K63 Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.

Article Snippet: K48 Tetra-Ubiquitin (Cat# SBB-UP0070) and K63 Tetra-Ubiquitin (Cat# SBB-UP0073) were a product of South Bay Bio.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Knock-Out, Mutagenesis

( a ) A scatter plot of caspase-4 interactors with altered affinity for caspase-4 in the presence of UBXN1. HEK293T cells were primed with IFN-γ for 12 h, transfected with a FLAG-CASP4 and vector or Myc-UBXN1 plasmid for 24 h. Immunoprecipitation (IP) was performed with an anti-FLAG antibody and bound proteins were identified by mass spectrometry. A Log 2 foldchange (FC) represents the ratio of average precursor intensity of a caspase-4 bound protein in the FLAG-CASP4 + Myc-UBXN1 to FLAG-CASP4 + vector group. Proteins involved in ubiquitination are labeled. Dots represent mean of 3 independent experiments, p <L0.05 by two-tailed unpaired Student’s t-test with Benjamini–Hochberg. ( b ) The heatmap of binding Z-scores of caspase-4-binding, ubiquitination-related proteins from ( a ). ( c ) The frequency of covalent ubiquitination of individual caspase-4 lysine (K) residues. IP and MS were performed exactly as in ( a ). The ubiquitination ratio of a K is expressed as percent (%) of modified peptide (di-glycine residue) over total peptide counts containing the corresponding K. Error bar, mean ± S.E.M., N=3 independent experiments, no significance by two-way ANOVA, Bonferroni post-hoc test. ( d ) IP of FLAG-caspase-4 from HEK293T cells treated as in ( a ). Samples were immunoblotted (IB) with an anti-K48 Ub linkage specific antibody (left) and anti-FLAG M2 antibody (right). The arrowhead indicates unmodified FLAG-caspase-4. ( e ) The workflow to distinguish free polyubiquitin chain binding to caspase-4/11 from covalent ubiquitination of caspase-4/11. USP5: recombinant ubiquitin-specific proteinase 5 protein. Created with BioRender.com. ( f, g ) The IB of indicated proteins from ( e ), WCL, whole cell lysate. ( h ) Immunoblots of caspase-4/11 and GSDMD from HeLa and primary BMDMs. Cells were primed with IFN-γ, lysed, treated with Re-LPS in the presence (+) or absence (-) of a recombinant USP5 protein for 4 h. PBS, phosphate-buffered saline; FL, full-length; NT, cleaved N-terminal fragment. ( i ) Immunoblot of GSDMD in HeLa cells treated as in ( h ) without USP5. KO, knockout of UBXN1 . ( j ) Immunoblots of caspase-4 and GSDMD in HeLa cells treated as in ( h ) except that the recombinant USP5 was replaced by an USP5 inhibitor. ( k ) The schematic workflow for in vitro protein-protein interaction. Created with BioRender.com. K48-, K63-Ub 4 are E. coli -derived recombinant tetra ubiquitin chains. Recombinant 6xHis-UBXN1 (rUBXN1) is E. coli- derived too. ( l ) The immunoblots of indicated proteins in the eluates from ( k ). ( m ) In vitro caspase-4 activity assay with a fluorogenic substate, purified insect-derived caspase-4, E. coli -derived 6xHis-UBXN1 and Ub 4 , and Ra-LPS. Data are presented in mean + S.E.M., N=3 biological replicates, ** p <0.01, **** p <0.0001 by repeated measures two way-ANOVA, Sidak’s multiple comparisons test.

Journal: bioRxiv

Article Title: Unanchored ubiquitin chains promote the non-canonical inflammasome via UBXN1

doi: 10.1101/2024.10.30.621131

Figure Lengend Snippet: ( a ) A scatter plot of caspase-4 interactors with altered affinity for caspase-4 in the presence of UBXN1. HEK293T cells were primed with IFN-γ for 12 h, transfected with a FLAG-CASP4 and vector or Myc-UBXN1 plasmid for 24 h. Immunoprecipitation (IP) was performed with an anti-FLAG antibody and bound proteins were identified by mass spectrometry. A Log 2 foldchange (FC) represents the ratio of average precursor intensity of a caspase-4 bound protein in the FLAG-CASP4 + Myc-UBXN1 to FLAG-CASP4 + vector group. Proteins involved in ubiquitination are labeled. Dots represent mean of 3 independent experiments, p

Article Snippet: K48 Tetra-Ubiquitin (Cat# SBB-UP0070) and K63 Tetra-Ubiquitin (Cat# SBB-UP0073) were a product of South Bay Bio.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Mass Spectrometry, Labeling, Two Tailed Test, Binding Assay, Modification, Residue, Recombinant, Western Blot, Saline, Knock-Out, In Vitro, Derivative Assay, Activity Assay, Purification

( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and K63 Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.

Journal: bioRxiv

Article Title: Unanchored ubiquitin chains promote the non-canonical inflammasome via UBXN1

doi: 10.1101/2024.10.30.621131

Figure Lengend Snippet: ( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and K63 Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.

Article Snippet: K48 Tetra-Ubiquitin (Cat# SBB-UP0070) and K63 Tetra-Ubiquitin (Cat# SBB-UP0073) were a product of South Bay Bio.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Knock-Out, Mutagenesis

( a ) A scatter plot of caspase-4 interactors with altered affinity for caspase-4 in the presence of UBXN1. HEK293T cells were primed with IFN-γ for 12 h, transfected with a FLAG-CASP4 and vector or Myc-UBXN1 plasmid for 24 h. Immunoprecipitation (IP) was performed with an anti-FLAG antibody and bound proteins were identified by mass spectrometry. A Log 2 foldchange (FC) represents the ratio of average precursor intensity of a caspase-4 bound protein in the FLAG-CASP4 + Myc-UBXN1 to FLAG-CASP4 + vector group. Proteins involved in ubiquitination are labeled. Dots represent mean of 3 independent experiments, p <L0.05 by two-tailed unpaired Student’s t-test with Benjamini–Hochberg. ( b ) The heatmap of binding Z-scores of caspase-4-binding, ubiquitination-related proteins from ( a ). ( c ) The frequency of covalent ubiquitination of individual caspase-4 lysine (K) residues. IP and MS were performed exactly as in ( a ). The ubiquitination ratio of a K is expressed as percent (%) of modified peptide (di-glycine residue) over total peptide counts containing the corresponding K. Error bar, mean ± S.E.M., N=3 independent experiments, no significance by two-way ANOVA, Bonferroni post-hoc test. ( d ) IP of FLAG-caspase-4 from HEK293T cells treated as in ( a ). Samples were immunoblotted (IB) with an anti-K48 Ub linkage specific antibody (left) and anti-FLAG M2 antibody (right). The arrowhead indicates unmodified FLAG-caspase-4. ( e ) The workflow to distinguish free polyubiquitin chain binding to caspase-4/11 from covalent ubiquitination of caspase-4/11. USP5: recombinant ubiquitin-specific proteinase 5 protein. Created with BioRender.com. ( f, g ) The IB of indicated proteins from ( e ), WCL, whole cell lysate. ( h ) Immunoblots of caspase-4/11 and GSDMD from HeLa and primary BMDMs. Cells were primed with IFN-γ, lysed, treated with Re-LPS in the presence (+) or absence (-) of a recombinant USP5 protein for 4 h. PBS, phosphate-buffered saline; FL, full-length; NT, cleaved N-terminal fragment. ( i ) Immunoblot of GSDMD in HeLa cells treated as in ( h ) without USP5. KO, knockout of UBXN1 . ( j ) Immunoblots of caspase-4 and GSDMD in HeLa cells treated as in ( h ) except that the recombinant USP5 was replaced by an USP5 inhibitor. ( k ) The schematic workflow for in vitro protein-protein interaction. Created with BioRender.com. K48-, K63-Ub 4 are E. coli -derived recombinant tetra ubiquitin chains. Recombinant 6xHis-UBXN1 (rUBXN1) is E. coli- derived too. ( l ) The immunoblots of indicated proteins in the eluates from ( k ). ( m ) In vitro caspase-4 activity assay with a fluorogenic substate, purified insect-derived caspase-4, E. coli -derived 6xHis-UBXN1 and Ub 4 , and Ra-LPS. Data are presented in mean + S.E.M., N=3 biological replicates, ** p <0.01, **** p <0.0001 by repeated measures two way-ANOVA, Sidak’s multiple comparisons test.

Journal: bioRxiv

Article Title: Unanchored ubiquitin chains promote the non-canonical inflammasome via UBXN1

doi: 10.1101/2024.10.30.621131

Figure Lengend Snippet: ( a ) A scatter plot of caspase-4 interactors with altered affinity for caspase-4 in the presence of UBXN1. HEK293T cells were primed with IFN-γ for 12 h, transfected with a FLAG-CASP4 and vector or Myc-UBXN1 plasmid for 24 h. Immunoprecipitation (IP) was performed with an anti-FLAG antibody and bound proteins were identified by mass spectrometry. A Log 2 foldchange (FC) represents the ratio of average precursor intensity of a caspase-4 bound protein in the FLAG-CASP4 + Myc-UBXN1 to FLAG-CASP4 + vector group. Proteins involved in ubiquitination are labeled. Dots represent mean of 3 independent experiments, p

Article Snippet: K48 Tetra-Ubiquitin (Cat# SBB-UP0070) and K63 Tetra-Ubiquitin (Cat# SBB-UP0073) were a product of South Bay Bio.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Mass Spectrometry, Labeling, Two Tailed Test, Binding Assay, Modification, Residue, Recombinant, Western Blot, Saline, Knock-Out, In Vitro, Derivative Assay, Activity Assay, Purification