Journal: The EMBO Journal

Article Title: LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF‐κB to the nucleus

doi: 10.15252/embj.2022112006

Figure Lengend Snippet: A, B A fraction of OTULIN is localized at mitochondria where it suppresses M1‐linked ubiquitination. HEK293T cells were transfected with control or OTULIN siRNA. Cells were harvested 72 h (A) or 48 h (B) after transfection or 15 min after TNF treatment (25 ng/ml). Mitochondria were isolated by differential centrifugation and purified by ultracentrifugation using an OptiPrep™ density gradient. 38% (A) or 20% (B) of the mitochondrial fractions and 2% (A, B) of whole cell lysates were analyzed by immunoblotting using the antibodies indicated. For the detection of M1‐linked ubiquitin chains, either the 1E3 (A) or the 1F11/3F5/Y102L (B) antibody was used. C TNF induces M1‐ubiquitination at mitochondria in various cell types. The indicated cell types were treated with TNF (25 ng/ml, 15 min) and purified mitochondrial fractions analyzed as described in (A). D TNF‐induced mitochondrial M1‐ubiquitination does not occur in HOIP‐deficient cells. Wildtype (WT) and HOIP‐KO HAP cells were treated with TNF (25 ng/ml, 15 min) and analyzed as described in (A). E Mitochondria and M1‐linked ubiquitin co‐localize after TNF treatment. SH‐SY5Y cells were treated with TNF (25 ng/ml, 15 min), fixed, stained with antibodies against HSP60 (green) and M1‐ubiquitin (1E3, red), and analyzed by SR‐SIM. Scale bar, 5 μm. F TNF induces recruitment of LUBAC components to mitochondria. HEK293T cells were treated with TNF (25 ng/ml, 15 min) and analyzed as described in (A). G TNF induces a fast and transient increase in M1‐ and K63‐specific ubiquitination at mitochondria. HEK 293T cells were treated with TNF (25 ng/ml) for the indicated time and the mitochondrial fractions were analyzed by immunoblotting using M1‐, K63‐, and K48‐specific ubiquitin antibodies. Source data are available online for this figure.

Article Snippet: Tetra‐ubiquitin (linear) , Enzo Life Sciences , Cat# BML‐UW0785‐0100.

Techniques: Transfection, Isolation, Centrifugation, Purification, Western Blot, Staining

Journal: The EMBO Journal

Article Title: LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF‐κB to the nucleus

doi: 10.15252/embj.2022112006

Figure Lengend Snippet: A, B PINK1 is stabilized by TNF treatment. HEK293T cells were treated with TNF (25 ng/ml, 15 min) or CCCP (10 μM, 90 min) before harvesting or incubated with OTULIN‐specific siRNA for 48 h (A). Purified mitochondrial fractions were analyzed by immunoblotting using the indicated antibodies. Quantification of PINK1‐specific signals normalized to TIM23 is shown in the right panel. Data represent the mean values with standard deviations of four independent experiments. * P < 0.05. A two‐tailed nonparametric Mann–Whitney U ‐test was used to analyze statistical significance. In (A), *unprocessed PINK1; **processed PINK1. C TNF‐induced PINK1 stabilization does not induce mitophagy. HeLa cells expressing mt‐mKeima were treated with TNF (25 ng/ml) for 30 min or 16 h. As a control, mt‐mKeima ‐expressing Hela cells were treated with antimycin A and oligomycin (A/O, 10 μM each) for 1 h. The analysis was done by flow cytometry gating lysosomal and neutral mt‐mKeima. D Catalytically active HOIP ubiquitinates overexpressed PINK1. HEK293T cells were transfected with the plasmids indicated. After 24 h, the cells were harvested under denaturing conditions and PINK1 was immunoprecipitated via the V5 tag followed by immunoblotting against ubiquitin. E Recombinant HOIP ubiquitinates PINK1 in vitro . V5‐tagged‐PINK1 immunoprecipitated from transiently transfected HEK293T cells via the V5 tag was incubated with recombinant mouse Ube1, UBE2L3, C‐terminal HOIP, and ubiquitin for in vitro ubiquitination. The samples were then analyzed by immunoblotting using V5 antibodies. F Endogenous PINK1 is modified with M1‐ubiquitin chains after TNF treatment. HEK293T cells were treated with TNF (25 ng/ml, 15 min), then endogenous PINK1 was immunoprecipitated from mitochondrial fractions. Cells mildly overexpressing PINK1‐V5 were also included (lanes 4, 5, 8), to make sure that the immunoreactive bands seen for endogenous PINK1 indeed correspond to PINK1. As a control for the presence of M1‐linked ubiquitin chains, immunoprecipitated PINK1 was treated with recombinant OTULIN. As controls for the specificity of the immunoprecipitation, beads only (lanes 6–8) and beads plus IgG (lane 9) were included. The samples were analyzed by immunoblotting using antibodies against PINK1, M1‐ubiquitin, and phosphorylated ubiquitin. For immunoprecipitation of overexpressed PINK1, only 50% of cells were used in comparison to the immunoprecipitation of endogenous PINK1. Source data are available online for this figure.

Article Snippet: Tetra‐ubiquitin (linear) , Enzo Life Sciences , Cat# BML‐UW0785‐0100.

Techniques: Incubation, Purification, Western Blot, Two Tailed Test, MANN-WHITNEY, Expressing, Flow Cytometry, Transfection, Immunoprecipitation, Recombinant, In Vitro, Modification

Journal: The EMBO Journal

Article Title: LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF‐κB to the nucleus

doi: 10.15252/embj.2022112006

Figure Lengend Snippet: A, C Schematic representation of the HOIP constructs used to map the interaction between HOIP and PINK1. All constructs are equipped with an N‐terminal HA tag. IBR, in‐between RING domain; LDD, linear ubiquitin chain determining domain; NZF, nuclear protein localization 4‐type zinc finger domain; PUB, peptide N‐glycosidase/ubiquitin‐associated domain; RING, really interesting new gene; UBA, ubiquitin‐associated domain; ZF, zinc finger domain. B PINK1 co‐immunoprecipitates with the N‐terminal part of HOIP. HEK293T cells were transfected with V5‐tagged PINK1 and the indicated HA‐tagged HOIP constructs. Twenty‐four hours after transfection cells were lysed under native conditions and subjected to immunoprecipitation using HA antibodies. Immunopurified proteins were analyzed by immunoblotting using V5 antibodies to detect PINK1. D The UBA domain enhances the interaction between HOIP and PINK1. HEK293T cells were analyzed as described in B. E PINK1 co‐elutes with HOIP in cellular lysates. HEK293T cells expressing V5‐tagged PINK1 were lysed, and soluble proteins were separated by size‐exclusion chromatography. Fractions were collected and analyzed by immunoblotting using antibodies against HOIP and V5. F PINK1 phosphorylates M1‐linked tetra‐ubiquitin in vitro . M1‐linked tetra‐ubiquitin was incubated with or without recombinant tcPINK1 in kinase buffer for 48 h. The samples were analyzed by Phos‐tag™ SDS–PAGE and immunoblotting using ubiquitin antibodies. G The efficiency of OTULIN to hydrolyze M1‐linked ubiquitin is decreased in the presence of PINK1. Recombinant M1‐linked tetra‐ubiquitin was incubated with or without recombinant Tribolium castaneum tcPINK1 for 48 h. Then, recombinant OTULIN was added for the indicated time. The reaction was stopped by adding Laemmli sample buffer and the samples were analyzed by immunoblotting using ubiquitin antibodies.

Article Snippet: Tetra‐ubiquitin (linear) , Enzo Life Sciences , Cat# BML‐UW0785‐0100.

Techniques: Construct, Transfection, Immunoprecipitation, Western Blot, Expressing, Size-exclusion Chromatography, In Vitro, Incubation, Recombinant, SDS Page

Journal: The EMBO Journal

Article Title: LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF‐κB to the nucleus

doi: 10.15252/embj.2022112006

Figure Lengend Snippet: PINK1 antagonizes OTULIN activity in cells. HEK293T cells expressing HOIP and HOIL‐1L were transfected with PINK1, WT OTULIN, or the inactive OTULIN mutant W96A, as indicated. The cells were lysed 24 h later under denaturing conditions, and lysates were subjected to affinity purification using the Strep‐tagged UBAN domain of NEMO to enrich proteins modified with M1‐linked ubiquitin. Proteins affinity‐purified by Strep‐Tactin beads were immunoblotted against ubiquitin. Catalytically active PINK1 increases p‐S65‐ubiquitin at mitochondria. HEK293T cells were transfected with wildtype PINK1 or kinase‐dead (K/D) PINK1. Forty‐eight hours after transfection, the cells were treated with TNF (25 ng/ml, 15 min) and lysed. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against p‐S65‐ubiquitin, PINK1, and VDAC (loading control). The TNF‐induced increase in p‐S65‐ubiquitin is abolished in PINK1‐deficient cells. HEK293T cells were transfected with control or PINK1‐specific siRNA and treated with TNF (25 ng/ml, 15 min) or CCCP (10 μM, 90 min) 48 h after transfection. The whole cell lysates were analyzed by immunoblotting using the indicated antibodies. TNF‐induced M1‐ubiquitination is reduced in PINK1‐deficient cells. WT and PINK1‐KO MEFs were treated with TNF (25 ng/ml, 15 min) and then harvested. Purified mitochondrial fractions were subjected to affinity purification using the Strep‐tagged UBAN domain, as described in (A), followed by immunoblotting using M1‐ubiquitin‐specific antibodies, Strep‐Tactin conjugated to horse radish peroxidase (to control the UBAN pulldown efficiency), and TIM23 (input control). Quantification of the M1‐ubiquitin‐positive signal intensities is shown in the lower panel. Data represent the mean values with standard deviations of three independent experiments; n.s., not significant, *** P < 0.001. A two‐way ANOVA test was used to analyze statistical significance. Source data are available online for this figure.

Article Snippet: Tetra‐ubiquitin (linear) , Enzo Life Sciences , Cat# BML‐UW0785‐0100.

Techniques: Activity Assay, Expressing, Transfection, Mutagenesis, Affinity Purification, Modification, Purification, Western Blot

Journal: The EMBO Journal

Article Title: LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF‐κB to the nucleus

doi: 10.15252/embj.2022112006

Figure Lengend Snippet: A STS‐induced mitochondrial Bax recruitment is reduced by TNF. HeLa cells were treated with STS (1 μM, 1 h) with or without a 15 min pretreatment with TNF (25 ng/ml) and then harvested. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against Bax and M1‐ubiquitin. The input was immunoblotted for TIM23 (upper panel). Bax‐specific signal intensities were quantified and normalized to TIM23‐specific signals (lower panel). Data represent the mean with standard error of eight independent experiments. * P < 0.05, *** P < 0.001. Kruskal–Wallis test followed by Dunn's multiple comparison test, n = 8. B STS‐induced cytochrome c release is decreased by TNF. HeLa cells were treated as described in (A). The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies. GAPDH was used as a reference. Quantification of five biological replicates is shown in the lower panel. Signal intensities were quantified and normalized to that of GAPDH. * P < 0.05. Kolmogorow–Smirnov normality test, paired t ‐test, two‐tailed, n = 5. C, D TNF treatment prevents damage of mitochondrial cristae under proapoptotic conditions. (C) SH‐SY5Y cells were treated as described in (A), fixed and embedded and the mitochondrial ultrastructure was imaged by electron microscopy. Scale bar: 400 nm. (D) Cristae abundance (average cristae number per mitochondrial area) and cristae length (average cristae length per mitochondrium) were analyzed by Imaris 9.8. * P < 0.05, ** P < 0.01, *** P < 0.01. Cristae abundance: Shapiro–Wilk normality test, One‐way ANOVA followed by Tukey's multiple comparison test, n = 27. Cristae length: Kruskal–Wallis test followed by Dunn's multiple comparison test, n = 24–27. Bars represent mean ± SEM. E The fast anti‐apoptotic effect of TNF is not affected by the NF‐κB inhibitor IκBα. SH‐SY5Y cells were transiently transfected with the NF‐κB super‐repressor IκBα‐2S or luciferase as a control. Twenty‐four hours later, the cells were treated with STS (5 μM, 2 h) with or without a 15 min pretreatment with TNF (25 ng/ml). Cells were fixed and stained by antibodies against active caspase‐3. Signal intensities were quantified by immunocytochemistry ancf IκBα‐2S was tested by immunoblotting using antibodies against the HA tag. Data represent the standard deviation of three independent experiments; at least 300 cells were counted per experiment. * P < 0.05, ** P < 0.01. Student's t ‐test, two‐tailed, n = 3. F The protective effect of TNF is dependent on HOIP expression. HeLa cells were transiently transfected with control or HOIP siRNA. Forty‐eight hours after transfection, cells were treated with STS (1 μM, 1 h) with or without a 15 min TNF pretreatment (25 ng/ml) and then harvested. The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies. HOIP silencing efficiency was analyzed in whole cell lysates using antibodies against HOIP. GAPDH and actin were immunoblotted as input controls. G The protective effect of TNF is dependent on PINK1 expression. HeLa cells were transiently transfected with control or PINK1 siRNA and treated as described in (F). The cytosolic fractions were analyzed by immunoblotting using cytochrome c and PINK1 antibodies. GAPDH was immunoblotted as input control. Source data are available online for this figure.

Article Snippet: Tetra‐ubiquitin (linear) , Enzo Life Sciences , Cat# BML‐UW0785‐0100.

Techniques: Purification, Western Blot, Two Tailed Test, Electron Microscopy, Transfection, Luciferase, Staining, Immunocytochemistry, Standard Deviation, Expressing

Journal: The EMBO Journal

Article Title: LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF‐κB to the nucleus

doi: 10.15252/embj.2022112006

Figure Lengend Snippet: Nuclear translocation of p65 is impaired in cells silenced for PINK1 expression. SH‐SY5Y cells were transfected with control or PINK1‐specific siRNAs. For rescue experiments, cells were co‐transfected with wildtype PINK1, PINK1Δ77, or kinase‐dead PINK1‐K/D. Two days after transfection the cells were treated with TNF (25 ng/ml, 25 min), fixed, and stained with p65 antibodies. The fraction of cells showing nuclear translocation of p65 was determined for each condition. Data represent the mean ± SEM of three independent experiments. The experiment was performed in triplicates and more than 300 cells were quantified per experiment and condition. *** P < 0.001, Student's t ‐test, two‐tailed, n = 3. PINK1 phosphorylates ubiquitinated NEMO. HEK293T cells were co‐transfected with V5‐tagged wildtype PINK1 or kinase‐dead PINK1‐K/D and HA‐tagged NEMO. One day after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using p‐S65‐ubiquitin antibodies. The input was immunoblotted for NEMO, PINK1, p‐S65‐ubiquitin, and GAPDH. Linear ubiquitination of NEMO is reduced in PINK1‐deficient cells. HEK293T cells were co‐transfected with HA‐NEMO and control or PINK1‐specific siRNAs. Forty‐eight hours after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using M1‐ubiquitin antibodies. The input was immunoblotted for NEMO and actin. PINK1 silencing efficiency was determined by real‐time RT–PCR. Bars represent mean ± SD with three technical replicates. Source data are available online for this figure.

Article Snippet: Tetra‐ubiquitin (linear) , Enzo Life Sciences , Cat# BML‐UW0785‐0100.

Techniques: Translocation Assay, Expressing, Transfection, Staining, Two Tailed Test, Immunoprecipitation, Western Blot, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF‐κB to the nucleus

doi: 10.15252/embj.2022112006

Figure Lengend Snippet:

Article Snippet: Tetra‐ubiquitin (linear) , Enzo Life Sciences , Cat# BML‐UW0785‐0100.

Techniques: Recombinant, Luciferase, In Situ

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3

doi: 10.1073/pnas.1710866114

Figure Lengend Snippet: SH3P2 binds ubiquitin conjugates. (A) Schematic presentation of full-length SH3P2(FL) and truncation constructs SH3P2(BAR) and SH3P2(SH3) used in C and D. (B) Tetra-ubiquitin (UB)–binding assay with recombinant MBP-SH3P2. Equal amounts of bead-bound MBP-SH3P2(FL) and linear (L), K48-linked (K48), or K63-linked (K63) tetra-UB were incubated together. After washing, the binding of tetra-UB to MBP-SH3P2(FL) was analyzed by immunoblotting with anti-UB (P4D1) and anti-MBP antibodies. Empty amylose beads were used as a negative control. (C) Tetra-UB–binding assay with GST-SH3P2(SH3) and GST-SH3P2(BAR). Equal amounts of bead-bound GST-SH3P2(SH3), GST-SH3P2(BAR), and GST were incubated with L, K48, or K63 tetra-UB. After washing, the binding of tetra-UB to the GST-fusion proteins was analyzed by immunoblotting with anti-UB (P4D1) and anti-GST antibodies. (D) UB-binding assay with GST-SH3P2(SH3) and GST. Bead-bound recombinant GST-SH3P2(SH3) and GST were incubated with crude extracts of amsh1-1. After washing, bead-bound materials were subjected to immunoblotting with anti-UB (P4D1) and anti-GST antibodies. The arrowhead indicates the position of GST-SH3P2(SH3).

Article Snippet: For in vitro-binding assays, Pierce Glutathione Magnetic Beads saturated with 25, 50, or 65 pmol of GST-fusion proteins were incubated with an equimolar amount of another protein or tetra-ubiquitin chains (Enzo Life Science) in 400 μL of cold buffer (50 mM Tris⋅HCl at pH 7.5, 150 mM NaCl, 10 mM MgCl 2 , 0.05% Tween-20) under rotation at 4 °C.

Techniques: Construct, Binding Assay, Recombinant, Incubation, Western Blot, Negative Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3

doi: 10.1073/pnas.1710866114

Figure Lengend Snippet: The vps23.1-3−/−vps23.2-1+/− seedlings show severe developmental defects and accumulate ubiquitylated proteins. (A) Phenotypes of vps23.1-3−/−vps23.2-1+/− seedlings. Photographs of 7-d-old wild type (WT; Left), vps23.1-3−/−vps23.2-1+/− (Center), and vps23.1-3−/−vps23.2-1+/− harboring the amish3p2 construct (Right) are shown. (Right Inset) Among the vps23.1-3−/−vps23.2-1+/−amish3p2 seedlings, seedlings with tricotyledons were also found. (Scale bars: 5 mm; Inset, 0.5 mm.) (B) Accumulation of ubiquitin (UB) conjugates in vps23 mutants. Total protein extracts of 7-d-old WT, vps23.1-3−/−vps23.2-1+/− WT-looking progenies (WT-like siblings), and aberrant vps23.1-3−/−vps23.2-1+/− (small) seedlings were subjected to immunoblotting using an anti-UB antibody. Note that vps23.1-3−/−vps23.2-1+/− (small) seedlings accumulate ubiquitylated proteins compared with the WT seedlings. Anti-CDC2 antibody was used as a loading control. (C) Possible function of SH3P2 on CCVs. SH3P2 can bind to the K63-linked ubiquitin chain of a cargo protein at the plasma membrane (PM) and, through its BAR domain, can bind to the membrane during clathrin-mediated endocytosis of ubiquitylated cargo proteins. SH3P2 could recruit ESCRT-I to the CCV via the interaction with the subunit VPS23. VPS23 binds the ubiquitin chain of the endocytosed cargo and leads the cargo to the downstream ESCRT machinery. The DUB AMSH3 can bind both the SH3P2 and the ESCRT-I subunit VPS23 and could be recruited by both proteins to the CCVs. Deubiquitylated membrane cargos might be recycled back to the PM.

Article Snippet: For in vitro-binding assays, Pierce Glutathione Magnetic Beads saturated with 25, 50, or 65 pmol of GST-fusion proteins were incubated with an equimolar amount of another protein or tetra-ubiquitin chains (Enzo Life Science) in 400 μL of cold buffer (50 mM Tris⋅HCl at pH 7.5, 150 mM NaCl, 10 mM MgCl 2 , 0.05% Tween-20) under rotation at 4 °C.

Techniques: Construct, Western Blot

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

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

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

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