k63 linked ubiquitin  (Novus Biologicals)

Journal: Research

Article Title: USP53 Drives Ethanol-Induced Myocardial Injury by Promoting K63 Deubiquitination-Dependent RIPK1 Activation at K377

doi: 10.34133/research.0823

Figure Lengend Snippet: USP53 deubiquitinates K63-linked ubiquitin chain of RIPK1 at K377 promoting RIPK1 phosphorylation and CM injury. (A) Ubiquitination of immunoprecipitated RIPK1 in AC16 cells transfected with USP53-siRNA or CTRL. (B) Exogenous ubiquitination assays of HA-RIPK1 in HEK-293T cells cotransfected with Myc-Ub, HA-RIPK1, and Flag-USP53 or Flag-USP53-C41S. (C) Exogenous ubiquitination assays of HA-RIPK1 in HEK-293T cells cotransfected with HA-RIPK1 and ubiquitin variants, including Myc-Ub-M1, Myc-Ub-K11, Myc-Ub-K48, or Myc-Ub-K63 in the presence of Flag-USP53 or Flag-USP53-C41S. (D) K63 ubiquitination of immunoprecipitated RIPK1 was analyzed by Western blotting in AC16 cells transfected with USP53-siRNA. (E) Western blotting of markers in necroptosis and apoptosis pathways of USP53-deficient AC16 cells under EtOH treatment, with or without USP53-C41S reconstitution ( n = 3 each). (F) Schematic diagram of ubiquitination sites on the ID of RIPK1 identified from PhosphosSitePlus ( www.phosphosite.org ). (G) Exogenous ubiquitination assays of lysine residue mutants of RIPK1 in HEK-293T cells. HEK-293T cells were cotransfected with Myc-Ub and lysine residue mutants of RIPK1 in the presence of Flag-USP53 or Flag-USP53-C41S. (H) Western blotting analysis of pRIPK1 Ser 166 in AC16 cells transfected with RIPK1 lysine residue mutants ( n = 3 each). (I) TUNEL staining of EtOH-stimulated AC16 cells cotransfected with Flag-USP53 and RIPK1 or RIPK1-K377R mutant ( n = 3 each) (scale bars, 50 μm).

Article Snippet: Primary antibodies used included USP53 (Immunoway, YT4845), p-RIPK1 (Ser 166 ; Proteintech, 28252-1-AP), RIPK1 (CST, 3493T), p-RIPK3 (Ser 227 , Abcam, ab209384; Ser 232 , Abcam, ab195117), RIPK3 (CST, 10188T), p-MLKL (Ser 358 , Abcam, ab187091; Ser 345 , Abcam, ab196436), MLKL (Immunoway, YM8455), cleaved caspase-3 (CST, 9661T), caspase-3 (CST, 9662T), EGR1 (CST, 4153T), ubiquitin (CST, 20326T), K63-linked ubiquitin (CST, 5621T), K48-linked ubiquitin (CST, 8081T), HA (CST, 3724T), Flag (CST, 14793T), MYC (CST, 2276T), cleaved GSDMD (Abcam, ab215203), GSDMD (Proteintech, 20770-1-AP), ACSL4 (Proteintech, 22401-1-AP), GPX4 (CST, 59735T), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, 60004-1-AP).

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Immunoprecipitation, Transfection, Western Blot, Residue, TUNEL Assay, Staining, Mutagenesis

Journal: Nature Communications

Article Title: USP37 prevents unscheduled replisome unloading through MCM complex deubiquitination

doi: 10.1038/s41467-025-59770-7

Figure Lengend Snippet: a USP37 was depleted using siRNA for 48 h in RPE1 cells stably expressing a 6xHis-FLAG-tagged ubiquitin construct. Ubiquitinated proteins were pulled down using Ni-NTA, revealing that USP37 siRNA increases endogenous MCM7 ubiquitination, as observed by immunoblotting. Representative of two biological replicates. b MCM7 ubiquitination was analyzed as described in ( a ), except that cells were treated with 5 µM of the p97i CB-5083 for the last 4 h before harvesting. Inhibition of p97 strongly increases MCM7 ubiquitination, and this is even more pronounced after USP37 depletion. Representative of three biological replicates. c HeLa cells stably expressing CDC45 GFP were depleted of USP37 before being stabilized prior to S phase using thymidine. After release, cells were treated with DMSO or 5 µM of p97i before CMG complexes were isolated using biotinylated anti-GFP Nanobodies (Nb) conjugated to Strep-tacin resin. Inhibition of p97 combined with depletion of USP37 significantly enhanced ubiquitination of endogenous MCM7. Representative of two biological replicates. d Ubiquitinated MCM7 isolated from HEK-293T cells was mixed with 100 nM of recombinant USP37 WT or a catalytically inactive mutant (C350S). The in vitro deubiquitination assay shows that USP37 WT, but not C350S, deubiquitinates Ub-MCM7. Representative of > three biological replicates. e FLAG-tagged USP37 was ectopically expressed for 48 hours and subsequently purified from HEK-293T cells by FLAG immunoprecipitation. USP37 immunoprecipitates were mixed with 1 µM of K11, K48, or K63 tetra-ubiquitin chains, revealing that USP37 cleaves Tetra- and Tri-Ub more efficiently than Di-Ub. Representative of two biological replicates.

Article Snippet: In parallel, K11, K48, or K63 Tetra-ubiquitin chains (UC-45, UCB-210, UC-310, R&D Systems) were prepared in DUB buffer at a final concentration of 2 μM, then mixed with the USP37 immunoprecipitates.

Techniques: Stable Transfection, Expressing, Ubiquitin Proteomics, Construct, Western Blot, Inhibition, Isolation, Recombinant, Mutagenesis, In Vitro, Purification, Immunoprecipitation

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Antibody panels used for immunohistochemistry on HD tissue microarrays.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Immunohistochemistry, Ubiquitin Proteomics

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Immunohistochemical profiling of HTT inclusion body ubiquitination and associated triage protein binding in the HD human middle temporal gyrus. Multiplexed immunohistochemical approaches were used to identify HTT inclusion bodies, ubiquitin species, and triage proteins in neurologically normal and HD human middle temporal gyrus tissue microarray cores. Example images from HD case, HC150, are shown. HTT inclusion body antibodies, EM48 ( A ), EPR ( B ), and MW1 ( C ), were used for labelling together with antibodies for pan-ubiquitin ( D ), K48- and K63-linked polyubiquitination ( E and F ), p62 ( G ), and ubiquilin 2 ( H ), with a Hoechst nuclear counterstain ( I ); scale bars = 20 μm.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Immunohistochemical staining, Ubiquitin Proteomics, Protein Binding, Microarray

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: HTT inclusion bodies are not frequently ubiquitinated, but when ubiquitinated, are predominantly ubiquitinated by K63-linked ubiquitin. Immunohistochemical labelling revealed that EM48, EPR, and/or MW1 HTT inclusion bodies were ubiquitinated by K48- and/or K63-linked ubiquitin ( A ); a representative image of K48- and K63-ubiquitinated HTT inclusion bodies from HD case, HC145, is shown; scale bars = 10 μm. The ubiquitination status of each HTT inclusion body was determined by labelling for pan-, K48-, and K63-linked ubiquitin, where positive labelling was identified if the maximum intensity was above manually determined thresholds. The percentage of EM48 + versus EM48- ( B ), EPR + versus EPR- ( C ), and MW1 + versus MW1- ( D ) HTT inclusion bodies that were ubiquitinated (either pan, K48-, and/or K63-linked) were compared using a Wilcoxon matched-pairs signed rank test. The percentage of ubiquitinated HTT inclusion bodies was determined for each EM48, EPR, and MW1 +/- phenotype per HD case ( E ), and compared between phenotypes using a mixed-effects analysis, with Geisser-Greenhouse correction and Tukey’s multiple comparisons test. The percentage of ubiquitinated HTT inclusion bodies ubiquitinated by K48- versus K63-linked ubiquitin was compared using a Wilcoxon matched-pairs signed rank test ( F ). The percentage of ubiquitinated EM48 + versus EM48- ( G ), EPR + versus EPR- ( H ), and MW1 + versus MW1- ( I ) HTT inclusion bodies ubiquitinated by K48- versus K63-linked ubiquitin were compared using an ordinary two-way ANOVA with Tukey’s multiple comparisons test. The percentage of EM48, EPR, and MW1 +/- immunophenotypes HTT inclusion bodies identified as being ubiquitinated by K48- or K63-linked chains were compared using an ordinary two-way ANOVA with Sidak’s multiple comparisons test ( J ). Data are presented as truncated violin plots ( n = 20). Statistical significance of differences shown for B-D and F-J: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Statistical significance for E is shown in Supplementary Table 3.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Ubiquitin Proteomics, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Summary of HTT inclusion body characteristics. Heatmap organised by HTT inclusion body phenotype, with each column representing a single case and each bar coloured according to that case’s value for the characteristic outlined by the row title ( A ). Schematic illustrating the general characteristics of each HTT inclusion body phenotype: (1) EPR + MW1 + inclusion bodies are more frequently located in the nucleus compared to other phenotypes, (2) HTT inclusion bodies that label for more than one epitope-specific antibody are more frequently ubiquitinated, and that ubiquitination occurs more frequently by K63- compared to K48-linked ubiquitin chains, (3) Ubiquitinated HTT inclusion bodies are more frequently tagged by ubiquilin 2 than p62 ( B ). Schematic summarising our hypothesis of HTT inclusion body immunophenotype, ubiquitination, and triage protein tagging with increasing HD severity ( C ); created in BioRender.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Ubiquitin Proteomics