pcdna5 frt to myc p31 comet  (Addgene inc)

Journal: Cell & Bioscience

Article Title: Linear ubiquitination of p31 comet by HOIP couples cytokine response with mitotic regulation

doi: 10.1186/s13578-025-01416-8

Figure Lengend Snippet: HOIP specifically binds to p31 comet . A . Immunoblot (IB) analysis of whole-cell lysates (WCL) and anti-Flag immunoprecipitates (IP) derived from asynchronous 293 T cells transfected with Flag-HOIP and the indicated HA-tagged constructs. EV: empty vector. B . IB analysis of WCL, anti-HA, and anti-Flag IP derived from asynchronous 293 T cells transfected with HA-p31 comet and the indicated Flag-tagged constructs. C . Domain structures of p31 comet and HOIP. The ubiquitinated lysines are indicated in the p31 comet domain structure. Functional domains are indicated in the HOIP domain structure. Catalytically inactive HOIP mutants are indicated at the bottom. D . IB analysis of WCL, anti-HA, and anti-Flag IP derived from asynchronous 293 T cells transfected with HA-p31 comet and the indicated Flag-HOIP constructs. E . IB analysis of WCL and anti-Flag IP derived from asynchronous 293 T cells transfected with Flag-HOIP and the indicated HA-p31 comet constructs. F . p31 comet bound to HOIP in vitro. Immunopurified Flag-HOIP was incubated with the indicated recombinant GST proteins before washing and being resolved by SDS-PAGE and IB. G . Immunofluorescent staining of HeLa cells using anti-HOIP and anti-p31 comet antibodies, DAPI was used for DNA staining. Representative images shown here are from a mitotic HeLa cell. (See also Figure S2)

Article Snippet: pcDNA5 FRT TO myc p31 Comet , Addgene , 59,833.

Techniques: Western Blot, Derivative Assay, Transfection, Construct, Plasmid Preparation, Functional Assay, In Vitro, Incubation, Recombinant, SDS Page, Staining

Journal: Cell & Bioscience

Article Title: Linear ubiquitination of p31 comet by HOIP couples cytokine response with mitotic regulation

doi: 10.1186/s13578-025-01416-8

Figure Lengend Snippet: HOIP promotes linear ubiquitination of p31 comet . A. Immunoblot (IB) analysis of whole cell lysates (WCL) and Ni–NTA (Ni-nitrilotriacetic acid) affinity precipitates derived from 293T cells transfected with HA-p31 comet , Flag-HOIP and the indicated His-UB constructs. The indicated lysine residue denotes that all but the indicated lysine (K) was mutated to arginine (R). K0: all the seven lysines of the ubiquitin were mutated to arginine. B . IB analysis of WCL and Ni–NTA affinity precipitates derived from 293T cells transfected with HA-p31 comet , Flag-HOIP and the indicated His-UB constructs. K0-ΔGG denotes the K0-UB with the two C-terminal glycine (G) residues deleted. C . IB analysis of WCL and Ni–NTA affinity precipitates derived from 293T cells transfected with HA-p31 comet , His-UB, and the indicated Flag-HOIP constructs. D . Bacterially purified recombinant His-p31 comet proteins were incubated with E1, E2, ubiquitin (UB), immune-purified WT, and catalytic inactive (R2-3 CS + HA) Flag-HOIP as indicated at 30 °C for 60 min before resolved by SDS-PAGE and IB analysis. E . IB analysis of WCL and Ni–NTA affinity precipitates derived from control (Ctrl) and sgHOIP-293T cells transfected with HA-p31 comet and His-UB constructs as indicated. 293T cells were treated with 300 nM nocodazole for 16 h. Mitotic cells were collected by mechanical shake-off. F . Crystal structure of p31 comet (PDBID 2QYF) was superimposed with the AlphaFold predicted structure to highlight the localization of the C-terminal lysine residues. G . IB analysis of WCL and Ni–NTA affinity precipitates derived from 293 T cells transfected with Flag-HOIP, His-UB, and the indicated HA-p31 comet constructs. (See also Figure S3)

Article Snippet: pcDNA5 FRT TO myc p31 Comet , Addgene , 59,833.

Techniques: Ubiquitin Proteomics, Western Blot, Derivative Assay, Transfection, Construct, Residue, Purification, Recombinant, Incubation, SDS Page, Control

Journal: Cell & Bioscience

Article Title: Linear ubiquitination of p31 comet by HOIP couples cytokine response with mitotic regulation

doi: 10.1186/s13578-025-01416-8

Figure Lengend Snippet: Delayed mitotic exit is observed in HOIP-depleted cells. A–B Immunoblot (IB) analysis of whole-cell lysates (WCL) derived from control (sgGFP) or HOIP-deleted (sgHOIP) HeLa ( A ) and HCT116 ( B ) cells. Cells were arrested in mitosis using 300 nM nocodazole for 16 h. Mitotic cells were collected by mechanical shake-off and released back into the cell cycle in media without nocodazole. Cells were harvested at the indicated time points after nocodazole release. C–D . IB analysis of WCL derived from HCT116 ( C ) and HeLa ( D ) cells transduced with shScr (as the negative control) or the indicated lentiviral sh p31 constructs. E. IB analysis of WCL derived derived from control (sgGFP) or p31 comet -deleted (sgp31) HCT116 cells. Cells were arrested in mitosis using 300 nM nocodazole for 16 h. Mitotic cells were collected by mechanical shake-off and released back into the cell cycle in media without nocodazole. Cells were harvested at the indicated time points after nocodazole release. F–G. Time-lapse microscopy of sgGFP and sgHOIP HeLa cells stably expressing a H2B-YFP retroviral construct. F Representative images showing that sgHOIP-HeLa took a significantly longer time to complete the mitosis. G Quantification of the recorded mitotic duration times (minutes) of sgGFP and sgHOIP cells, n = 200, Student’s t-test. H–I. Time-lapse microscopy of sgGFP and sgp31 H2B-YFP-HeLa cells. H Representative images showing that sgp31-HeLa took a significantly longer time to complete the mitosis. I Quantification of the recorded mitotic duration times (minutes) of sgGFP and sgHOIP cells, n = 50, Student’s t-test. (See also Figure S4)

Article Snippet: pcDNA5 FRT TO myc p31 Comet , Addgene , 59,833.

Techniques: Western Blot, Derivative Assay, Control, Transduction, Negative Control, Construct, Time-lapse Microscopy, Stable Transfection, Expressing, Retroviral

Journal: Cell & Bioscience

Article Title: Linear ubiquitination of p31 comet by HOIP couples cytokine response with mitotic regulation

doi: 10.1186/s13578-025-01416-8

Figure Lengend Snippet: Cells expressing a linear ubiquitination-deficient p31 comet exhibit prolonged mitosis. A. Immunoblot (IB) analysis of whole cell lysates (WCL) and anti-ubiquitin immunoprecipitates (IP) derived from HEK293 cells. Cells were treated with 50 ng∙ml −1 TNFα for 10 min as indicated before harvest. B . IB analysis of WCL and anti-HA IP derived from HEK293 cells. C . IB analysis of WCL and anti-HA IP derived from HEK293 cells. Cells were treated with 50 ng∙ml −1 TNFα for 10 min as indicated before harvest. D . IB analysis of WCL and anti-HA IP derived from 293 T cells transfected with Myc-p31 comet , HA-PLK1, His-UB and Flag-HOIP as indicated. E–F . Time-lapse microscopy of sgp31-H2B-YFP-HeLa cells stably expressing WT or 5KR-p31 comet . E Representative images showing that 5KR-p31-HeLa cells took a significantly longer time to complete the mitosis. F Quantification of the recorded mitotic duration times (minutes) of WT- and 5KR-p31 comet expressing cells, n = 50, Student’s t-test. (See also Figure S5)

Article Snippet: pcDNA5 FRT TO myc p31 Comet , Addgene , 59,833.

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Derivative Assay, Transfection, Time-lapse Microscopy, Stable Transfection

Journal: Cell & bioscience

Article Title: Linear ubiquitination of p31 comet by HOIP couples cytokine response with mitotic regulation.

doi: 10.1186/s13578-025-01416-8

Figure Lengend Snippet: Fig. 5 Cells expressing a linear ubiquitination-deficient p31comet exhibit prolonged mitosis. A. Immunoblot (IB) analysis of whole cell lysates (WCL) and anti-ubiquitin immunoprecipitates (IP) derived from HEK293 cells. Cells were treated with 50 ng∙ml−1 TNFα for 10 min as indicated before harvest. B. IB analysis of WCL and anti-HA IP derived from HEK293 cells. C. IB analysis of WCL and anti-HA IP derived from HEK293 cells. Cells were treated with 50 ng∙ml−1 TNFα for 10 min as indicated before harvest. D. IB analysis of WCL and anti-HA IP derived from 293 T cells transfected with Myc-p31comet, HA-PLK1, His-UB and Flag-HOIP as indicated. E–F. Time-lapse microscopy of sgp31-H2B-YFP-HeLa cells stably expressing WT or 5KR-p31comet. E Representative images showing that 5KR-p31-HeLa cells took a significantly longer time to complete the mitosis. F Quantification of the recorded mitotic duration times (minutes) of WT- and 5KR-p31comet expressing cells, n = 50, Student’s t-test. (See also Figure S5)

Article Snippet: HOIL-1L MilliporeSigma clone 2E2 MABC576 TUBULIN MilliporeSigma T-5168 VINCULIN MilliporeSigma V-4505 Flag (polyclonal) MilliporeSigma F-2425 Flag (monoclonal) MilliporeSigma F-3165 anti-Flag agarose beads MilliporeSigma A-2220 anti-HA agarose beads MilliporeSigma A-2095 peroxidase-conjugated anti-mouse secondary antibody MilliporeSigma A-4416 peroxidase-conjugated anti-rabbit secondary antibody MilliporeSigma A-4914 Plasmids pCMV3flag8HOIP Addgene 50,015 pCMV3flag8HOIL-1L Addgene 50,016 pCMV3-flag8-SHARPIN Addgene 50,014 pcDNA5 FRT TO myc p31 Comet Addgene 59,833 pERB160-hMAD1 Addgene 58,285 pcDNA5-EGFP-AIDBubR1 Addgene 47,330 VL3 (Flag-Mad2) Addgene 16,047 pcDNA3 HA human NEMO Addgene 13,512 pLKO.1-puro Addgene 8453 lentiCRISPRv2-puro Addgene 98,290 pHK-mTRIP13 Dr. Kevin D. Corbett, UCSD pcDNA3-6His-ubiquitin and mutants PMID: 31,015,455

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Derivative Assay, Transfection, Time-lapse Microscopy, Stable Transfection

Journal: PLoS Pathogens

Article Title: Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling

doi: 10.1371/journal.ppat.1005346

Figure Lengend Snippet: (A) BGLF4 induced phosphorylation of proteins involved in SAC (spindle assembly checkpoint) activation and APC/C (anaphase promoting complex /cyclosome) inhibition. P: phosphorylation; Ac: acetylation. (B) Western blot analysis showing co-precipitation of BGLF4 with MPS1, CDC20 and p31 Comet but not BUB1. BGLF4 was co-transfected with MPS1, BUB1, CDC20 or p31 Comet in 293T cells as indicated. Cell lysates were immunoprecipitated with anti-HA antibody, anti-Myc or IgG control followed by immunoblotting with anti-BGLF4, anti-HA, anti-Flag or anti-Myc antibodies as indicated. (C) BGLF4 phosphorylates TIP60, MPS1, PP1α and CDC20. (Upper) Purified wild-type (WT) and kinase-dead (KD) BGLF4 were mixed with GST-TIP60 (aa 1–290), GST-MPS1 (a.a. 410–517), GST-PP1α, GST-CDC20 and GST. Autoradiography showing only WT BGLF4 phosphorylates TIP60, MPS1, PP1α and CDC20. GST-TIP60 (a.a. 1–290) and GST were included as positive and negative controls respectively. Arrows indicate the positions of the phosphorylated substrates. (Lower) Immunoblot showing purified WT and KD BGLF4 proteins (Elute) used in the assay. Arrows indicate the positions of purified untagged BGLF4 (Elute) and Halo-BGLF4 proteins in the total cell lysate (Lysate). See also .

Article Snippet: HA-cdc20 was a gift from Marc Kirschner (Addgene plasmid # 11594)[ ]. pcDNA5 FRT TO myc p31 Comet was a gift from Stephen Taylor (Addgene plasmid # 59833) [ ].

Techniques: Phospho-proteomics, Activation Assay, Inhibition, Western Blot, Transfection, Immunoprecipitation, Control, Purification, Autoradiography