Journal: Nature Communications

Article Title: USP44 regulates irradiation-induced DNA double-strand break repair and suppresses tumorigenesis in nasopharyngeal carcinoma

doi: 10.1038/s41467-022-28158-2

Figure Lengend Snippet: a SDS-PAGE of HA-immunoprecipitated proteins separated from SUNE1 cells stably overexpressing HA-USP44. Red lines indicate the proteins of interest. b Co-IP with anti-HA or anti-FLAG antibody in SUNE1 cells revealed the exogenous association of USP44 and Ku80. c Immunofluorescence staining revealed the cellular location of exogenous HA-USP44 (green) and endogenous Ku80 (red) at 0.5 h after exposure to 6Gy IR. Scale bars, 10 μm. d USP44 inhibited Ku80 protein expression but not its mRNA expression in a dose-dependent manner. e The effect of CHX treatment and greyscale analysis of the results in 293T cells transfected with FLAG- Ku80 and HA- USP44 or the empty vector plasmids, as well as in sgNC or sg USP44 SUNE1 cells. f The effect of MG132 and CQ treatment in 293T cells transfected with the indicated plasmids. g HEK293T cells transfected with FLAG- Ku80 , HA- Ub and HA- USP44 or the empty plasmids were subjected to denature-IP and immunoblotted with the indicated antibodies. h Co-IP assay detecting the exogenous association of USP44 and TRIM25 and the endogenous association of USP44, TRIM25 and Ku80 in NPC cells. i TRIM25 inhibited Ku80 protein expression but not its mRNA expression in a dose-dependent manner. j The effect of MG132 and CQ treatment in 293T cells transfected with FLAG- Ku80 and FLAG- TRIM25 or the empty vector plasmids. k The effect of CHX treatment and greyscale analysis of the results in 293T cells transfected with FLAG- Ku80 and MYC- TRIM25 or the empty vector plasmids, as well as in sgNC or sg TRIM25 SUNE1 cells. l , m HEK293T cells transfected with the indicated plasmids or siRNAs were subjected to denature-IP and then immunoblotted with the indicated antibody. Data in d , e and i , k are presented as the mean ± SD; the P values were determined using the two-tailed Student’s t -test; n = 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: HEK293T cells obtained from the American Type Tissue Culture Collection (ATCC) were cultured in DMEM (Invitrogen) supplemented with 10% FBS.

Techniques: SDS Page, Immunoprecipitation, Stable Transfection, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Expressing, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: USP44 regulates irradiation-induced DNA double-strand break repair and suppresses tumorigenesis in nasopharyngeal carcinoma

doi: 10.1038/s41467-022-28158-2

Figure Lengend Snippet: a USP44 promoted Ku80 protein expression but not its mRNA expression in a dose-dependent manner. b , c The effect of CHX ( b ), MG132 and CQ ( c ) treatment in 293T cells transfected with FLAG- TRIM25 and HA- USP44 or the empty vector plasmids, as well as in sgNC or sg USP44 SUNE1 cells. d , e HEK293T cells transfected with HA- USP44 or the empty vector ( d ) and sgNC or sg USP44 SUNE1 cells ( e ) co-transfected with FLAG- TRIM25 or MYC- TRIM25 and a vector encoding HA-WT-Ub or its mutants (HA-K48O-Ub or HA-K63O-Ub) were subjected to denature-IP and immunoblotted with the indicated antibodies. f HEK293T and NPC cells transfected with vector plasmid, HA-USP44 or HA-USP44 (C282A) were immunoblotted with the indicated antibodies. g HONE1 cells transfected with the vector plasmid, HA- USP44 or HA- USP44 (C282A) together with MYC- TRIM25 and HA-K48O- Ub were subjected to denature-IP and immunoblotted with the indicated antibodies. h Mass spectrometry analysis of TRIM25 ubiquitination sites. i HEK293T cells were transfected with the vector plasmid or HA- USP44 , HA- Ub and Flag- TRIM25 WT or KR mutants, subjected to denature-IP with anti-Flag beads and then analysed by immunoblot with an anti-HA or anti-Flag antibody. j SUNE1 and HONE1 cells exposed to IR (6Gy) transfected with the indicated plasmids and siRNAs were fixed 0.5 h later and co-immunostained with the anti-Ku80 antibody. Scale bars, 10 μm. Data in a and b are presented as the mean ± SD; the P values were determined using the two-tailed Student’s t -test); n = 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: HEK293T cells obtained from the American Type Tissue Culture Collection (ATCC) were cultured in DMEM (Invitrogen) supplemented with 10% FBS.

Techniques: Expressing, Transfection, Plasmid Preparation, Mass Spectrometry, Ubiquitin Proteomics, Western Blot, Two Tailed Test

Journal: Oncotarget

Article Title: Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription

doi:

Figure Lengend Snippet: A. Empty vector (EV) or 3xFLAG-CASZ1b construct was transiently transfected into HEK293T cells, and anti-FLAG antibody was used for co-immunoprecipitation (co-IP) of the CASZ1b complex from the whole cell extracts. EV transfected cell extracts were used as control. The co-IP products were resolved by SDS-PAGE and stained with simply blue safe stain reagent. The lanes were sectioned and digested with trypsin. Extracted peptides were sequenced by mass spectrometry. B. EV and 1xFLAG-CASZ1b construct was transiently transfected into HEK293T cells. The cell extracts were used for co-IP with anti-FLAG antibody, and probed for subunit of NuRD and histone H3 by western blot (left panel), or probed for DNA repair proteins by western blot (right panel). C. Tetracycline inducible FLAG-CASZ1b construct was stably transfected into SY5Y cells, then the cells were cultured with or without 1 μg/μl tetracycline for 24 hr. The cell extracts were used for co-IP with anti-FLAG antibody, and probed for indicated proteins by western blot. D. Tetracycline inducible FLAG-CASZ1b construct was stably transfected into SY5Y cells, then the cells were cultured with or without 1 μg/μl tetracycline for 24 hr. The cell extracts were used for co-IP with anti-FLAG antibody, and probed for indicated proteins by western blot.

Article Snippet: Human embryonic kidney cells (HEK293T) were obtained from ATCC.

Techniques: Plasmid Preparation, Construct, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, SDS Page, Staining, Mass Spectrometry, Western Blot, Stable Transfection, Cell Culture

Journal: Oncotarget

Article Title: Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription

doi:

Figure Lengend Snippet: A. The HEK293T whole cell extracts were used for co-IP with MTA1 or PARP1 anti antibody, and probed for endogenous CASZ1 by western blot (left panel); The mouse ES cell extracts were used for co-IP with MTA1 or PARP1 anti antibody, and probed for endogenous CASZ1 by western blot (right panel). B. Mouse ES cell extracts were fractionated through a gel filtration column, and selected fractions were then resolved by SDS-PAGE and western blotting performed to indentify the indicated proteins.

Article Snippet: Human embryonic kidney cells (HEK293T) were obtained from ATCC.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Filtration, SDS Page

Journal: Oncotarget

Article Title: Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription

doi:

Figure Lengend Snippet: A. Cartoon of CASZ1b mutant constructs (ZF represents zinc finger), to the right of the cartoon, the relative transcriptional activity of CASZ1b mutants compared to wild type CASZ1b is shown. B. EV, FLAG-CASZ1b, or FLAG-CASZ1b mutant construct was transiently transfected into HEK293T cells, and anti-FLAG antibody was used for co-immunoprecipitation (co-IP) of the CASZ1b complex from the whole cell extracts. The co-IP products were resolved by SDS-PAGE and stained with comassie stain to show the pull down of CASZ1b and the mutant constructs, or probed for GAPDH by western blotting to show the specificity of co-IP. C. EV, FLAG-CASZ1b or the mutant construct was transiently transfected into HEK293T cells. The cell extracts were used for co-IP with anti-FLAG antibody, and probed for the subunits of NuRD by western blot. D. EV, FLAG-CASZ1b or the mutant construct was transiently transfected into HEK293T cells. The cell extracts were used for co-IP with anti-FLAG antibody, and probed for DNA repair proteins by western blot.

Article Snippet: Human embryonic kidney cells (HEK293T) were obtained from ATCC.

Techniques: Mutagenesis, Construct, Activity Assay, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, SDS Page, Staining, Western Blot

Journal: Oncotarget

Article Title: Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription

doi:

Figure Lengend Snippet: A. Cartoon of CASZ1b mutant constructs (ZF represents zinc finger), to the right of the cartoon, the relative transcriptional activity of CASZ1b mutants compared to wild type CASZ1b is shown. B. EV, FLAG-CASZ1b or the mutant construct was transiently transfected into HEK293T cells. The cell extracts were used for co-IP with anti-FLAG antibody, and probed for the subunits of NuRD by western blot. C. EV, FLAG-CASZ1b or the mutant construct was transiently transfected into HEK293T cells. The cell extracts were used for co-IP with anti-FLAG antibody, and probed for DNA repair proteins by western blot.

Article Snippet: Human embryonic kidney cells (HEK293T) were obtained from ATCC.

Techniques: Mutagenesis, Construct, Activity Assay, Transfection, Co-Immunoprecipitation Assay, Western Blot

Journal: Oncotarget

Article Title: Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription

doi:

Figure Lengend Snippet: A. a. Putative PAR binding motif existed in the middle of CASZ1b; b. this motif is conserved in different species; c. generate CASZ1b PAR binding motif mutant constructs. B. EV, FLAG-CASZ1b or the PAR binding mutant construct was transiently transfected into HEK293T cells. The cell extracts were used for co-IP with anti-FLAG antibody, and probed for PAR and PARP1 by western blot. C. Cartoon of CASZ1b N-terminus and PAR binding mutant constructs (ZF represents zinc finger), to the right of the cartoon, the transcriptional activity of CASZ1b mutants compared to wild type CASZ1b is shown. D. EV, FLAG-CASZ1b N-terminus mutant or PAR binding mutant construct was transiently transfected into HEK293T cells. The cell extracts were used for co-IP with anti-FLAG antibody, and probed for NuRD subunits, DNA repair proteins and histone H3 by western blot.

Article Snippet: Human embryonic kidney cells (HEK293T) were obtained from ATCC.

Techniques: Binding Assay, Mutagenesis, Construct, Transfection, Co-Immunoprecipitation Assay, Western Blot, Activity Assay

Journal: Oncotarget

Article Title: Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription

doi:

Figure Lengend Snippet: A. EV or FLAG-CASZ1b was transiently transfected into HEK293T cells with or without PARP inhibitor (AZD2281) treatment. The cell extracts were used for co-IP with anti-PARP1 antibody, and probed for PAR, PARP1 and CASZ1b by western blot. B. EV or FLAG-CASZ1b was transiently transfected into HEK293T cells with or without PARP inhibitor (AZD2281) treatment. The cell extracts were used for co-IP with anti-FLAG antibody, and probed for indicated proteins by western blot.

Article Snippet: Human embryonic kidney cells (HEK293T) were obtained from ATCC.

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot

Journal: Oncotarget

Article Title: Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription

doi:

Figure Lengend Snippet: A. EV, FLAG-CASZ1b N-terminus mutant or FLAG-CASZ1b PAR binding mutant constructs were transiently transfected into HEK293T cells, 24 hr the cells were harvested, and the mRNA level of CASZ1b target gene NGFR was evaluated by realtime PCR (data are represented as mean ± SD). The input levels of CASZ1b and mutants were shown by western blot. B. The alignment of the N-terminus of human CASZ1 (hCas) and zebra fish CASZ1 (zCas). C. Cartoon of Gal4DBD and CASZ1b fusion protein constructs. D. Gal4DBD-CASZ1b fusion constructs, 5xGal4 UAS luciferase reporter vector and beta-galactosidase reporter vector) were transiently transfected into HEK293T cells, and the expression of Gal4DBD-CASZ1b fusion protein was assessed by western blot. E. Gal4DBD-CASZ1b fusion constructs, 5xGal4 UAS luciferase reporter vector and beta-galactosidase reporter vector were transiently transfected into HEK293T cells for 24 hr, then treated with or without HDAC inhibitor (TSA) for another 26 hr, and the cells were harvested, and the luciferase activity was evaluated using luciferase assay reagent (data are represented as mean ± SD). F. Schematic map of CASZ1b protein to show NuRD, H3/DNA repair proteins binding sites.

Article Snippet: Human embryonic kidney cells (HEK293T) were obtained from ATCC.

Techniques: Mutagenesis, Binding Assay, Construct, Transfection, Western Blot, Luciferase, Plasmid Preparation, Expressing, Activity Assay

Journal: Nature biotechnology

Article Title: Proteome labelling and protein identification in specific tissues and at specific developmental stages in an animal

doi: 10.1038/nbt.2860

Figure Lengend Snippet: (a) Western blot analysis demonstrates the efficient amino acid dependant expression of an mCherry-EGFP fusion protein separated by an amber stop codon bearing a C-terminal HA-tag (mCh-TAG-EGFP-HA) in HEK293T cells. Anti-FLAG detected tagged PylRS (b) Specific labelling of mCh-TAG-EGFP-HA (immunoprecipitated from 106 cells) with 4a (20μM in 50μL PBS, 1h, RT) confirms the incorporation of 3 into protein in HEK293 cells. (c) SORT-M labelling of 3 that is statistically incorporated into newly synthesised proteins across the whole proteome of mammalian cells directed by six different PylRS/PyltRNAXXX mutants using 0.5 mM 3. Labeling with 4g (20μM in PBS, 1h, RT, as above). The amino acids in parentheses are the natural amino acids encoded by the endogenous tRNA bearing the corresponding anti-codon.

Article Snippet: Adherent HEK293T cells (ATCC CRL-11268; 4×10 6 per immunoprecipitation) were transfected with 7.5 μg p4CMVE-U6-PylT and 7.5ug pPylRS-mCherry-TAG-EFGP-HA 18 using TransIT-293 transfection reagent according to the manufacturer’s protocol and cultured for 48 hours in DMEM/10%FBS, supplemented with 0.5 mM 1 or 2 mM 3 where indicated.

Techniques: Western Blot, Expressing, Immunoprecipitation, Labeling

Journal: Nature communications

Article Title: Tumour suppressor TRIM33 targets nuclear β-catenin degradation

doi: 10.1038/ncomms7156

Figure Lengend Snippet: a, Co-IP of endogenous TRIM33 with β-catenin in U87/EGFRvIII cells ( left panel ) and DLD-1 cells ( right panel ). TRIM33 was immunoprecipitated, and the amount of TRIM33 bound to β-catenin was determined using an immunoblot with an anti-β-catenin antibody. b, Interaction of endogenous β-catenin ( top panel ) and endogenous TRIM33 ( bottom panel ) was induced upon Wnt3a treatment. IP was performed with whole-cell lysates of HEK 293T cells pretreated with 100 ng/ml Wnt3a. Whole-cell lysates were probed for input. The amounts of TRIM33 and β-catenin immunoprecipitated were normalized with IgG. c, The C-terminal domain of β-catenin mediates the interaction of this protein with TRIM33. Top panel, Schematic illustration of β-catenin. delC and delN constructs lack the C terminus and N terminus of the protein, respectively. Bottom panel, β-Catenin deletion mutants were co-expressed with FLAG-TRIM33 in 293T cells in the presence of MG132. The cells were subjected to IP with a FLAG antibody followed by immunoblotting (IB) with FLAG and Myc antibodies. Whole-cell lysates were directly subjected to IB using FLAG or Myc antibody as input.

Article Snippet: HEK 293T cells (the American Type Culture Collection, ATCC) and GSK-3β–knockout MEFs (gift from Dr. James R. Woodgett, Mount Sinai Hospital, Toronto); U87 (ATCC) and U87/EGFRvIII (gift from Dr. Oliver Bogler, MD Anderson Cancer Center, Houston) GBM cells; and DLD-1, SW480, and HT-29 colorectal cancer cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (HyClone).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Construct

Journal: Nature communications

Article Title: Tumour suppressor TRIM33 targets nuclear β-catenin degradation

doi: 10.1038/ncomms7156

Figure Lengend Snippet: a, GSK-3β −/− MEFs were transfected with the constructs indicated; 48 h later the cells were harvested and the nuclear extracts analysed by IB. b–d, HEK 293T cells were co-transfected with the indicated constructs; 48 h later the cells were harvested and the nuclear extracts analysed by IB. e , Top panel, Diagram of TRIM33 protein domains. The N-terminal domain (TRIM) consists of a RING-finger domain, two B-box domains (B) and a coiled-coil domain (CC); the C-terminal domain consists of plant homeodomain (PHD)/bromodomain (Br) regions. Both domains are coupled by a middle region. Bottom panel, FLAG-TRIM33 and β-catenin-Myc were co-expressed in HEK 293T cells in the presence of MG132. FLAG-TRIM33 protein was immunoprecipitated using an anti-FLAG antibody. β-Catenin–bound TRIM33 protein was detected using an immunoblot with an anti-Myc antibody. Whole-cell lysates were directly subjected to IB using anti-Myc antibody as input. f, U87/EGFRvIII cells were transfected with the indicated plasmids, the nuclear extracts were prepared, and the β-catenin level was examined. Lamin B was used as a loading control for nuclear fractions. g , HEK 293T cells were transfected with the indicated plasmids. Transfection efficiency was normalized by co-transfection with the pRL-TK plasmid. Luciferase activity was measured 36 h after transfection by the dual luciferase assay. Luciferase activity is shown as relative luciferase activity (RLA) compared to that in cells transfected with the control vector. h , HEK 293T cells were transfected with the indicated plasmids. Luciferase activity was measured 36 h after transfection. i , HEK 293T cells transfected with or without TRIM33 were transfected with TOP-FLASH or FOP-FLASH, which was followed by Wnt3a treatment for 4 h. j , U87 cells transfected with or without TRIM33 were transfected with TOP-FLASH or FOP-FLASH, which was followed by EGF treatment for 10 h. In g , h , i , and j , each error bar indicates the variation between the means of 3 independent experiments.

Article Snippet: HEK 293T cells (the American Type Culture Collection, ATCC) and GSK-3β–knockout MEFs (gift from Dr. James R. Woodgett, Mount Sinai Hospital, Toronto); U87 (ATCC) and U87/EGFRvIII (gift from Dr. Oliver Bogler, MD Anderson Cancer Center, Houston) GBM cells; and DLD-1, SW480, and HT-29 colorectal cancer cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (HyClone).

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, Control, Cotransfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Nature communications

Article Title: Tumour suppressor TRIM33 targets nuclear β-catenin degradation

doi: 10.1038/ncomms7156

Figure Lengend Snippet: a, HEK 293T cells with or without TRIM33 depletion by siRNA were transfected as indicated. Cells were treated with MG132 (10 μM for 4 h) prior to lysis and then subjected to anti-β-catenin IP followed by anti-HA with immunoblot analysis. b, HEK 293T cells with or without TRIM33 depletion by shRNA were transfected as indicated. Experiments were performed as described for a. c , Nuclear extracts of U87/EGFRvIII cells pretreated with MG132 (10 μM for 4 h) were immunoprecipitated. ΔRING, TRIM33 lacking the RING domain; CAmut, TRIM33 with point mutations (C125A/C128A) in the RING domain. d , In vivo ubiquitination assays performed in HEK 293T cells transiently transfected with HA-tagged K63-only ubiquitin or K48-only ubiquitin. e, TRIM33 ubiquitylation of β-catenin in vitro . Purified β-catenin was subjected to in vitro ubiquitylation by TRIM33 in the absence or presence of active PKCδ. β-Catenin was detected with anti-GST antibody. f & g, U87 cells with or without TRIM33 depletion were transfected with β-TrCP siRNA or control siRNA. Cells were treated for 8 h with 100 ng/ml Wnt-3a (Wnt-on) (f) or with 100 ng DKK1 (Wnt-off) (g). Experiments were performed as described for a.

Article Snippet: HEK 293T cells (the American Type Culture Collection, ATCC) and GSK-3β–knockout MEFs (gift from Dr. James R. Woodgett, Mount Sinai Hospital, Toronto); U87 (ATCC) and U87/EGFRvIII (gift from Dr. Oliver Bogler, MD Anderson Cancer Center, Houston) GBM cells; and DLD-1, SW480, and HT-29 colorectal cancer cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (HyClone).

Techniques: Transfection, Lysis, Western Blot, shRNA, Immunoprecipitation, In Vivo, Ubiquitin Proteomics, In Vitro, Purification, Control

Journal: Nature communications

Article Title: Tumour suppressor TRIM33 targets nuclear β-catenin degradation

doi: 10.1038/ncomms7156

Figure Lengend Snippet: a, HEK 293T cells were transfected with the indicated plasmids. Luciferase activity was measured 36 h after transfection. Each error bar indicates the variation between the means of 3 independent experiments. b, HEK 293T cells were transfected with FLAG-tagged TRIM33 and the indicated Myc-tagged β-catenin mutants. Whole-cell lysates were immunoprecipitated with anti-FLAG antibody, and immunocomplexes were probed with antibodies against the indicated proteins. CAmut, TRIM33 with point mutations (C125A/C128A) in the RING domain. c, HEK 293T cells were transfected with FLAG-tagged TRIM33 and the indicated Myc-tagged β-catenin mutants. Experiments were performed as described for b. d, Alignment of the amino acid regions containing the TRIM33 binding motif in β-catenin orthologues. e, β-Catenin S715A mutants had a longer protein half-life. Left panel, HEK 293T cells were transfected with the indicated mutants. Cells were incubated with cyclohexamide (CHX) for the indicated times, collected, and analysed by IB as indicated. Right panel, the amount of β-catenin (WT or mutants) is represented relative to the amount at time 0. f, HEK 293T cells were transfected with FLAG-TRIM33 alone or in combination with β-catenin WT –Myc or β-catenin–Myc serine substitution mutants. The transfected cells were treated with MG132 4 h prior to harvest, which was followed by IP with anti-Myc antibody. Cell lysates and the immunoprecipitates were analysed by IB with the indicated antibodies.

Article Snippet: HEK 293T cells (the American Type Culture Collection, ATCC) and GSK-3β–knockout MEFs (gift from Dr. James R. Woodgett, Mount Sinai Hospital, Toronto); U87 (ATCC) and U87/EGFRvIII (gift from Dr. Oliver Bogler, MD Anderson Cancer Center, Houston) GBM cells; and DLD-1, SW480, and HT-29 colorectal cancer cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (HyClone).

Techniques: Transfection, Luciferase, Activity Assay, Immunoprecipitation, Binding Assay, Incubation

Journal: Nature communications

Article Title: Tumour suppressor TRIM33 targets nuclear β-catenin degradation

doi: 10.1038/ncomms7156

Figure Lengend Snippet: a, HEK 293T cells transiently transfected with or without TRIM33 were transfected with the indicated constructs. Cells were pretreated with Bis-I (2 mM) or Go6976 (2 mM). Each error bar indicates the variation between the means of 3 independent experiments. b, U87/EGFRvIII cells were transiently transfected with constitutively active (+) or kinase-dead (−) PKC mutants. Nuclear cell lysates were collected at 48 h post-transfection and subjected to immunoblot analyses using the indicated antibodies. c, U87 cells with or without TRIM33 depletion were transfected with siRNAs targeting PKCδ or control siRNA, incubated with cyclohexamide (CHX) for the indicated times, and collected and analysed. Top panel , IB results. Bottom panel , Quantitation of nuclear β-catenin was normalized to the loading control and expressed relative to 0 h. d, PKCδ silencing inhibited TRIM33–β-catenin interaction. U87/EGFRvIII cells were transfected with either a siRNA targeting PKCδ or control siRNA; 48 h post-transfection, cells were collected and whole-cell lysates were immunoprecipitated and immunoblotted as indicated. e, PKCδ stimulated the binding of TRIM33 to β-catenin. U87 cells were transiently transfected with constitutively active (+) or kinase-dead (−) PKCδ. The transfected cells were treated with MG132 4 h prior to harvest. The experiment was performed as described in d. f & g, Localization of endogenous PKCδ in HEK 293T (f) and U87 (g) cells. Nuclei were visualized with DAPI (blue). Scale bars: 20 μm. h, In vitro kinase assays were performed with purified active PKCδ and purified β-catenin proteins and analysed by autoradiography. i, U87 cells were treated with Wnt3a (100 ng/ml) with or without MG132 for the indicated times. IB was performed with nuclear lysates of the cells with indicated antibody. j, U87 cells transfected with either PKCδ siRNA targeting or control siRNA were treated with Wnt3a (100 ng/ml) for 8 h. IB was performed with nuclear lysates of the cells with anti-pS715-β-catenin antibody.

Article Snippet: HEK 293T cells (the American Type Culture Collection, ATCC) and GSK-3β–knockout MEFs (gift from Dr. James R. Woodgett, Mount Sinai Hospital, Toronto); U87 (ATCC) and U87/EGFRvIII (gift from Dr. Oliver Bogler, MD Anderson Cancer Center, Houston) GBM cells; and DLD-1, SW480, and HT-29 colorectal cancer cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (HyClone).

Techniques: Transfection, Construct, Western Blot, Control, Incubation, Quantitation Assay, Immunoprecipitation, Binding Assay, In Vitro, Purification, Autoradiography

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 1. The CSFV NS5A interacts with NEMO. A, B HEK293T cells in 6-well plates were transfected with p3FLAG-NS5A or p3FLAG-CMV-10 (2.5 μg/well). At 48 h post-transfection, the cells were lysed for co-immunoprecipitation (co-IP) and Western blotting with indicated antibodies. C PK15 cells were infected with CSFV Shimen strain at an MOI of 1. At 48 hpi, the cells were lysed for co-IP and Western blotting with indicated antibodies.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Infection

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 2. Determination of the domain of NEMO required for the association with NS5A. A Schematic representation of NEMO mutant constructs. B HEK293T cells in 6- well plates were transfected with NEMO mutants (2.5 μg/well). After 48 h of transfection, the cells were lysed and incubated with Dynabeads™coupled with His-NS5A or His tag. The proteins co-purified with fusion protein His-NS5A or His tag and cell lysates were subjected to Western blotting analysis using the indicated antibodies.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Mutagenesis, Construct, Transfection, Incubation, Western Blot

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 3. Determination of the region of NS5A required for NS5A-NEMO interaction. A Schematic representation of NS5A mutant constructs. B HEK293T cells in 24-well plates were transfected with full-length NS5A or NS5A truncated mutant (1 μg/well). FLAG tag was used as control. After 48 h of transfection, the cells were lysed for co-immunoprecipitation and Western blotting assay with the indicated antibodies.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Mutagenesis, Construct, Transfection, FLAG-tag, Control, Immunoprecipitation, Western Blot

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 4. Determination of the region of NS5A required for inhibition of the NF-κB signaling. A–C HEK293T cells in 6-well plate were transfected with full-length NS5A or NS5A truncated mutant (2.5 μg/well). After 48 h, the cells were treated with or without TNF-α for 15 min and then harvested for detection of IKKα/β phos- phorylation (A), IκBα degradation (B), quantification of IFN-α mRNA (C). The expressing level of phosphorylated IKKα/β and IκBα was analyzed by Western blotting. IFN-α mRNA was detected by RT-PCR using the One-Step SYBR Prime Script RT-PCR kit. D HEK293T cells in 24-well plate were transfected with pNF-κB-Luc reporter (1 μg/well), PRL-TK internal control (0.5 μg/well). After 48 h, the cells were treated with or without TNF-α for 15 min and the NF-κB transcriptional activity was analyzed using the Dual-Lumi Luciferase Reporter Gene Assay Kit. E The expression level of NS5A and its truncated mutants in above experiments was detected by Western blotting. Data presented as means SD. Statistical analyses were performed using GraphPad Prism version 6.01 software (GraphPad Software, La Jolla, CA, USA).**, P < 0.01; ***, P < 0.001; NS, not significant.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Inhibition, Transfection, Mutagenesis, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Activity Assay, Luciferase, Reporter Gene Assay, Software

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 5. Reduction of NEMO induced by CSFV or NS5A. PK-15 cells (A) or PAM cells (B) were infected with CSFV Shimen strain at an MOI of 3 or 5. At 48 hpi, the cells were lysed for Western blotting analysis with the indicated antibodies. C, D HEK293T cells cultured in 6-well plates were transfected with the indicated vector (pcDNA-NEMO, 2.5 μg/well). At 48 h post-transfection, the cells were lysed for Western blotting analysis with the indicated antibodies. Data presented as means SD. Statistical analyses were performed using GraphPad Prism version 6.01 software (GraphPad Software, La Jolla, CA, USA).*, P < 0.05; **, P < 0.01.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Infection, Western Blot, Cell Culture, Transfection, Plasmid Preparation, Software

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 6. NS5A mediates proteasomal degradation of NEMO. HEK293T cells cultured in 6-well plates were co-transfected with pcDNA-NEMO and p3FLAG-NS5A vectors (2.5 μg/well) (A) or transfected with p3FLAG-NS5A vector alone (2.5 μg/well) (B). At 24 h post-transfection, the cells were incubated with MG132 for 24 h, then the cells were lysed for Western blotting analysis with the indicated antibodies. C Cell viability after MG132 treatment was evaluated by MTT assay. Data presented as means SD. Statistical analyses were performed using GraphPad Prism version 6.01 software (GraphPad Software, La Jolla, CA, USA). *, P < 0.05; **, P < 0.01; NS, not significant.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Cell Culture, Transfection, Plasmid Preparation, Incubation, Western Blot, MTT Assay, Software

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 7. NS5A mediates NEMO ubiquitination. PK-15 and HEK293T cells in 6-well plates were transfected with p3FLAG-NS5A or p3FLAG-CMV-10 (2.5 μg/well). At 24 h post-transfection, the cells were incubated with MG132 (10 μmol/L) for 24 h, and then the cells were lysed for immunoprecipitation with rabbit anti-NEMO mAb or nonspecific IgG. Western blotting analysis was performed by using mouse anti-Ub mAb, mouse anti-NEMO mAb and mouse anti-FLAG mAb.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Ubiquitin Proteomics, Transfection, Incubation, Immunoprecipitation, Western Blot

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 8. NS5A does not mediate K48-linked polyubiquitination of NEMO. PK-15 and HEK293T cells in 6-well plates were transfected with p3FLAG-NS5A or p3FLAG-CMV-10 (2.5 μg/well). At 24 h post-transfection, the cells were incubated with MG132 (10 μmol/L) for 24 h. Then the cells were lysed for immunopre- cipitation with rabbit anti-NEMO mAb, and Western blotting analysis was performed by using K48-linkage specific polyubiquitin (D9D5) rabbit mAb, mouse anti- NEMO mAb and mouse anti-FLAG mAb.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Transfection, Incubation, Western Blot

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 9. NS5A induces K27-linked polyubiquitination of NEMO. PK-15 (A and B) and HEK293T (C and D) cells in 6-well plates were transfected with p3FLAG-NS5A or p3FLAG-CMV-10 (2.5 μg/well). At 24 h post-transfection, the cells were incubated with MG132 (10 μmol/L) for 24 h. Then the cells were harvested for immunoprecipitation with rabbit anti-NEMO mAb and ubiquitination assay of NEMO with mouse anti-HA mAb.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Transfection, Incubation, Immunoprecipitation, Ubiquitin Proteomics

Journal: Virologica Sinica

Article Title: Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

doi: 10.1016/j.virs.2023.09.002

Figure Lengend Snippet: Fig. 10. NS5A inhibits K63-linked polyubiquitination of NEMO. PK-15 (A) and HEK293T (B) cells in 6-well plates were transfected with or without p3FLAG-NS5A or p3FLAG-CMV-10 vector (2.5 μg/well). Following incubation with MG132 (10 μmol/L) for 24 h and infection with SeV (500 HAU/well) for 12 h, the cells were lysed for immunoprecipitation with rabbit anti-NEMO mAb or nonspecific IgG, and Western blotting analysis was performed by using K63-linkage specific polyubiquitin (D7A11) rabbit mAb, and mouse anti-NEMO mAb or mouse anti-FLAG mAb.

Article Snippet: Briefly, DynabeadsTM (Thermo Fisher) with bound His-NS5A or His tag were incubated with the cell lysates from HEK293T cells expressing Myc-NEMOΔN1, Myc-NEMOΔN2, Myc-NEMOΔN3 or Myc-NEMOΔN1 for 10 min on a roller at 37 C. Proteins co-purified with His-NS5A or His-tagged protein were detected by western blotting with rabbit anti-Myc polyclonal antibody (Proteintech, China) for probing NEMO mutants and mouse anti-His tag mAb (Proteintech, China) for probing NS5A.

Techniques: Transfection, Plasmid Preparation, Incubation, Infection, Immunoprecipitation, Western Blot

Journal: iScience

Article Title: Dishevelled localization and function are differentially regulated by structurally distinct sterols

doi: 10.1016/j.isci.2025.112704

Figure Lengend Snippet: DVL2 subcellular localization is shifted in response to sterol change (A) Schematic illustrating the final steps of post-squalene cholesterol synthesis. Small molecule inhibitors are indicated in red, target proteins in blue. (B) Representative western blot of total DVL2 expression following inhibitor treatments. (C) Total cellular DVL2 normalized to GAPDH. N = 3 independent experiments. Data represent mean ± SEM. One-way ANOVA (F (4, 10) = 1.260 p = 0.347555) with Dunnett’s test. (D) Representative western blots of subcellular DVL2 following inhibitor treatments. (E) Subcellular DVL2 normalized to total actin. N = 3–6 independent experiments. Data represent mean ± SEM. One-way ANOVA with Dunnett’s test. (F) Confocal imaging of HEK293T-DVL2-mEGFP-KI cells following respective treatments. (G) Nuclear GFP/unit nuclear area. N = 48–55 individual cells. Data represent mean ± SEM. One-way ANOVA (F (3, 218) = 274.3, p < 0.000001) with Dunnett’s test. (H) Nuclear GFP normalized to total GFP/field. N = 3 independent experiments. One-way ANOVA (F (3, 8) = 62.68, p < 0.0001) with Dunnett’s test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 for all statistical tests. Scale bar = 25 μm.

Article Snippet: The following cell lines were utilized in this study: Human: HEK293T (ATCC, ACS-4500), HEK293T hDVL2-mGFP, HEK293T TCF/LEF-Luciferase (this manuscript), HEK293T minimal promoter-Luciferase (this manuscript); Mouse: L-Wnt-3A cells (ATCC, CRL-2647).

Techniques: Western Blot, Expressing, Imaging

Journal: iScience

Article Title: Dishevelled localization and function are differentially regulated by structurally distinct sterols

doi: 10.1016/j.isci.2025.112704

Figure Lengend Snippet: Nuclear DVL2 transport is FoxK2 dependent and sensitive to PDZ-domain inhibition (A) Co-IP between DVL2 and FoxK2 under sterol-impacted conditions. (B) Nuclear DVL2 binding to FoxK2. N = 3 independent experiments. Data represent mean ± SEM. One-way ANOVA with Dunnett’s test; F (2, 6) = 20.58, p = 0.0021 (nucleus); F (2, 6) – 11.22, p = 0.0094 (cytosol). (C) Model for NSC668036, FoxK2, and cholesterol binding to DVL2-PDZ. Predicted NSC668036 binding residues are green, cholesterol residues are purple, FoxK2 residues are cyan. (D) HEK293T-DVL2-mEGFP-KI cells following sterol treatments. (E) Nuclear GFP/unit nuclear area. N > 50 cells from 2 independent experiments. Data represent mean ± SEM. One-way ANOVA (F (4, 438) = 168.5, p < 0.0001) with Tukey’s test. (F) Nuclear GFP normalized to total GFP/field. N = 4 fields from 2 independent experiments. Data represent mean ± SEM. One-way ANOVA (F (4, 15) = 15.74, p < 0.0001) with Tukey’s test. (G) Co-IP between DVL2 and FoxK2. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 for all statistical tests. Scale bar = 25 μm. HC = heavy chain of IgG.

Article Snippet: The following cell lines were utilized in this study: Human: HEK293T (ATCC, ACS-4500), HEK293T hDVL2-mGFP, HEK293T TCF/LEF-Luciferase (this manuscript), HEK293T minimal promoter-Luciferase (this manuscript); Mouse: L-Wnt-3A cells (ATCC, CRL-2647).

Techniques: Inhibition, Co-Immunoprecipitation Assay, Binding Assay

Journal: iScience

Article Title: Dishevelled localization and function are differentially regulated by structurally distinct sterols

doi: 10.1016/j.isci.2025.112704

Figure Lengend Snippet: Loss of sterol homeostasis alters the DVL2 protein-protein interaction network (A) Venn diagram of DVL2-interacting proteins in HEK293T cells under sterol-specific conditions. (B) Representative key DVL2 interactions lost within LPDS treatment. (C) DVL2-Tp53 interaction network in FBS, LPDS, and AY9944 treatment. (D) Co-IP between endogenous DVL2 and p53 in HEK293T. (E) β-catenin and Axin1/2 interaction networks with DVL2 under sterol-specific conditions. (F) β-catenin and DVL2 interaction within the nucleus. (G) Co-IP of endogenous DVL2 and Axin2 in HEK293T cells. HC = heavy chain.

Article Snippet: The following cell lines were utilized in this study: Human: HEK293T (ATCC, ACS-4500), HEK293T hDVL2-mGFP, HEK293T TCF/LEF-Luciferase (this manuscript), HEK293T minimal promoter-Luciferase (this manuscript); Mouse: L-Wnt-3A cells (ATCC, CRL-2647).

Techniques: Co-Immunoprecipitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the E3 ubiquitin protein ligase ITCH diminishes binding to its cognate E2 ubiquitin ligase

doi: 10.1074/jbc.RA117.000408

Figure Lengend Snippet: Phosphorylation disrupts UbcH7 binding. A, the structure of the E6AP–UbcH7 complex (1C4Z) is shown to the left of the panel series with the E6AP HECT domain colored cyan and UbcH7 colored yellow. The upper panel highlights the E6AP–UbcH7 interface near E6AP residue Ser-638 at the H7 helix, which forms a side chain to main chain hydrogen bond with UbcH7 residue Phe-63 at the L1 loop. To model the ITCH–UbcH7 interface, the structure of the ITCH HECT domain (3TUG, colored green) is superimposed onto the E6AP HECT domain within the E6AP–UbcH7 complex and shown at the upper right panel. Similar to the E6AP residue Ser-638, the ITCH residue Ser-687 is predicted to form the same side chain to main chain hydrogen bond with the UbcH7 residue Phe-63. Mutation of Ser-687 to Asp (lower left) or phosphorylation of Ser-687 (lower right) would lead to loss of the hydrogen bond and significant steric hindrance at the E2–E3 interface, thus severely impairing ITCH–UbcH7 interaction. B, HEK 293T cells were transfected with FLAG-ITCH and FLAG-ITCH S687D alone or with Omni-UbcH7 for co-precipitation. FLAG-ITCH was immunoprecipitated (IP) in each sample. After stringent washing of IP samples, lysate and IP samples were ran on SDS-PAGE, transferred to nitrocellulose, and probed as indicated. Similar results were obtained in three independent experiments. C, HEK 293T cells were stimulated with 1 μg/ml TNFα for the indicated time points and UbcH7 was immunoprecipitated. After gentle washing, lysate and IP samples were processed by Western blotting. Membranes were probed as indicated. Similar results were obtained in two independent experiments. D, Biacore SPR sensorgrams for binding of UbcH7 to wildtype or S687D mutant ITCH.

Article Snippet: Cell culture, transient transfection, and immunoprecipitation/pulldown HEK 293T (CRL-1573; American Type Culture Collection) cells were grown in DMEM 10% SCS with 1% penicillin/streptomycin.

Techniques: Phospho-proteomics, Binding Assay, Residue, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Gentle, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the E3 ubiquitin protein ligase ITCH diminishes binding to its cognate E2 ubiquitin ligase

doi: 10.1074/jbc.RA117.000408

Figure Lengend Snippet: Phe-63 mediates the phosphorylation-dependent coupling between UbcH7 and ITCH. A, HEK 293T cells were transduced with lentivirus containing CRISPR-Cas9 with either a negative control-RNA guide or UbcH7 Guide 1-RNA. Lysate from pooled select UbcH7−/− clones was run next to controls on SDS-PAGE and probed for UbcH7 and GAPDH as shown. B, negative control and UbcH7−/− HEK 293T cells were transiently transfected with HA-ubiquitin, NTAP-RIPK2, and ITCH or ITCH C830A to assess ITCH ubiquitination of RIPK2. Cellular lysates were collected and cleared. Streptavidin beads were used to pull down RIPK2 via SBP tag within the NTAP tag. Samples were analyzed by Western blotting. C, UbcH7−/− HEK 293T cells were transiently transfected with HA-ubiquitin, NTAP-RIPK2, FLAG-ITCH, FLAG-ITCH C830A, and Myc-UbcH7. NTAP was precipitated under stringent washing conditions (1% SDS, 2 m NaCl). Samples were run on SDS-PAGE next to WT samples that were similarly transfected. Samples were subjected to Western blot analysis as indicated. Results shown are representative of three independent experiments. D, HA-tagged ubiquitin, NTAP-tagged RIPK2, and FLAG-ITCH were transiently transfected into UbcH7−/− HEK 293T cells with CRISPR-resistant UbcH7 WT, F63N, or F63S. RIPK2 was pulled down with streptavidin beads. Samples were washed and subjected to SDS-PAGE and immunoblotting as indicated.

Article Snippet: Cell culture, transient transfection, and immunoprecipitation/pulldown HEK 293T (CRL-1573; American Type Culture Collection) cells were grown in DMEM 10% SCS with 1% penicillin/streptomycin.

Techniques: Phospho-proteomics, Transduction, CRISPR, Negative Control, Clone Assay, SDS Page, Transfection, Ubiquitin Proteomics, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the E3 ubiquitin protein ligase ITCH diminishes binding to its cognate E2 ubiquitin ligase

doi: 10.1074/jbc.RA117.000408

Figure Lengend Snippet: UbcH7 deficiency phenocopies ITCH phosphorylation. A, UbcH7−/− HEK 293T cells were transiently transfected with HA-ubiquitin, NTAP-RIPK2, FLAG-ITCH, FLAG-ITCH S687A, Myc-UbcH7, or Myc-UBCH7 C86A as indicated. NTAP was precipitated under stringent washing conditions (1% SDS, 2 m NaCl). Western blotting was then performed with the indicated antibodies. Samples were subjected to Western blot analysis as indicated. Results shown are representative of three independent experiments. B, negative control and UbcH7−/− HEK 293T cells were stimulated with 10 ng/ml TNFα for the indicated time points. Lysates were standardized for total protein and analyzed and subjected to SDS-PAGE and Western blot analysis.

Article Snippet: Cell culture, transient transfection, and immunoprecipitation/pulldown HEK 293T (CRL-1573; American Type Culture Collection) cells were grown in DMEM 10% SCS with 1% penicillin/streptomycin.

Techniques: Phospho-proteomics, Transfection, Ubiquitin Proteomics, Western Blot, Negative Control, SDS Page

Journal: bioRxiv

Article Title: Increased ACTL6A Occupancy Within mSWI/SNF Chromatin Remodelers Drives Human Squamous Cell Carcinoma

doi: 10.1101/2021.03.22.435873

Figure Lengend Snippet: Increased expression of ACTL6A in SCCs drives ACTL6A occupancy within BAF complexes (A) Outline of method for quantifying the number of molecules of a specific protein per cell from different cell lines. (B) Quantifications of the number of ACTL6A molecules per cell compared to SMARCA4/SMARCA2. SCC cell lines: FaDu (head-and-neck), NCI-H520 (lung), and T.T (esophageal). KC: primary normal human keratinocytes. n=3 experiments. Error bars indicate SEM. * P < 0.05. (C) Co-immunoprecipitation (Co-IP) experiments using SMARCA4 antibody. Shown are Western blots and quantifications of relative levels of BAF subunits co-IP’d by SMARCA4 in SCC (FaDu) cells normalized to primary human keratinocytes (KC). n=3 experiments. Error bars indicate SEM. * P < 0.05. n.s.: not significant. (D) Co-IP experiments using SMARCA4 antibody in primary human keratinocytes (KC) transduced by lentivirus for ACTL6A over-expression and the vector control. Shown are Western blots and quantifications of relative levels of co-IP’d BAF subunits normalized to vector control. n=3 experiments. Error bars indicate SEM. * P < 0.05. n.s.: not significant. (E) Quantifications for co-immunoprecipitation (co-IP) experiments by SMARCA4 antibody. Relative levels of co-IP’d ACTL6A in ACTL6A-overexpressing condition normalized to vector-control condition by lentiviral transduction. FaDu: SCC cell line. KC: primary human keratinocytes. HEK293T: human embryonic kidney 293T cells. Error bars indicate SEM. * P < 0.05. n=2-3 experiments.

Article Snippet: HEK293T cells were purchased from Takara Bio USA (632180) and cultured in high glucose DMEM (GIBCO) medium supplemented with 10% FBS (GIBCO), 10 mM HEPES (Gibco), 1 mM sodium pyruvate (Gibco), 2mM GlutaMax (Gibco) and antibiotics.

Techniques: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Over Expression, Plasmid Preparation, Transduction

Journal: bioRxiv

Article Title: The E3 ubiquitin ligase MGRN1 targets melanocortin receptors MC1R and MC4R via interactions with transmembrane adapters

doi: 10.1101/2025.03.25.645338

Figure Lengend Snippet: (A) Schematic of the co-immunoprecipitation mass spectrometry (co-IP/MS) strategy used to identify MGRN1 interacting proteins. (B) Table listing nine proteins that interact exclusively with MGRN1 compared to wild-type controls. Peptide spectrum match (PSM). (C) Multiple sequence alignment (MSA) of the stem domain, transmembrane domain, cytoplasmic helix, and cytoplasmic tail of MEGF8, ATRN, and ATRNL1. MSA was performed using ClustalO and visualized with Jalview. Sequence identities between MEGF8 and ATRN/ATRNL1 were calculated using ClustalO. Secondary structure prediction of MEGF8 was generated with JPred, where helices are represented by red tubes and sheets by green arrows. (D, left) Schematic representation of the MOSMO-MEGF8-MGRN1 (MMM) and the ATRNL1-MGRN1 complexes. (D, right) Co-IP analysis of MEGF8 and ATRNL1 interactions. HEK293T cells were transiently transfected with 1D4-tagged MEGF8 or ATRNL1, FLAG-tagged MGRN1 (wild-type or catalytically inactive MGRN1 C279A/C282A ), and HA-tagged MOSMO. Proteins were immunoprecipitated using anti-1D4 beads and analyzed by western blot. MGRN1 interacts with both MEGF8 and ATRNL1, while MOSMO interacts specifically with only MEGF8. The asterisk (*) indicates a likely ATRNL1 dimer or oligomer that has been observed in previous studies . p38 serves as a loading control.

Article Snippet: The NIH/3T3 and HEK293T cells were passaged with 0.05% Trypsin-EDTA with phenol red (Gibco, Cat #25300062).

Techniques: Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Sequencing, Generated, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: The E3 ubiquitin ligase MGRN1 targets melanocortin receptors MC1R and MC4R via interactions with transmembrane adapters

doi: 10.1101/2025.03.25.645338

Figure Lengend Snippet: (A, Left) AlphaFold-predicted MC1R-MGRN1 and MC4R-MGRN1 interactions. (A and B) HEK293T cells were transfected with (A) MC1R-Venus or (B) MC4R-Venus along with either wild-type Mgrn1-Flag or the catalytically inactive Mgrn1 R318E -Flag linchpin mutant. We performed co-IP assays using anti-FLAG beads to pull down MGRN1 and then probed for either MC1R or MC4R using an anti-GFP antibody. In the absence of MGRN1-FLAG, MC1R and MC4R were not detected in the pulldown. However, both wild-type MGRN1 and MGRN1 R318E efficiently co-immunoprecipitated with MC1R and MC4R. (C and D) MC1R and MC4R ubiquitination were assessed after transient co-expression of the indicated proteins in HEK293T cells. Briefly, HEK293T cells were co-transfected with (C) MC1R-Venus or (D) MC4R-Venus along with HA-tagged ubiquitin ( HA-Ub ), FLAG-tagged Mgrn1 (wild-type or the Mgrn1 R318E linchpin mutant), and either the transmembrane adapter Megf8-1D4 or ATRNL1-1D4 . Cells were lysed under denaturing conditions, and MC1R or MC4R was purified by IP using GFP-Trap beads. The amount of HA-UB covalently conjugated to MC1R and MC4R was assessed using immunoblotting with an anti-HA antibody. GAPDH serves as a loading control. The asterisks (*) indicate the endogenous MGRN1.

Article Snippet: The NIH/3T3 and HEK293T cells were passaged with 0.05% Trypsin-EDTA with phenol red (Gibco, Cat #25300062).

Techniques: Transfection, Mutagenesis, Co-Immunoprecipitation Assay, Immunoprecipitation, Expressing, Purification, Western Blot, Control

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

Article Title: An AAGAB-to-CCDC32 handover mechanism controls the assembly of the AP2 adaptor complex.

doi: 10.1073/pnas.2409341121

Figure Lengend Snippet: Fig. 6. The α:σ2:CCDC32 complex sequentially recruits µ2 and β2 to complete AP2 assembly. (A) Diagrams of HA-tagged FL AP2 µ2 and β2 subunits and 3xFLAG- tagged AAGAB and CCDC32 used in co-IP experiments. (B and C) Representative immunoblots showing the interaction of 3xFLAG-tagged AAGAB and CCDC32 with HA-tagged µ2 (B) or β2 (C). The 3xFLAG-tagged AAGAB or CCDC32 protein was transiently expressed in HEK 293T cells with an empty vector (control) or plasmids encoding the indicated HA-tagged AP2 subunits. AAGAB and CCDC32 were immunoprecipitated using anti-FLAG antibodies, and proteins in the immunoprecipitates were detected using immunoblotting. (D) Representative immunoblots showing the interactions of µ2 and β2 subunits with the α:σ2:CCDC32 complex. His6-SUMO-tagged CCDC32 was coexpressed with GST-tagged α (trunk domain) and untagged σ2 in E. coli. The α:σ2:CCDC32 ternary complex was isolated from E. coli using glutathione beads and incubated with E. coli lysates containing His6-SUMO-tagged µ2 and β2 (trunk domain, a.a. 1–591) at 4 °C for the indicated periods. The glutathione beads were washed, and proteins bound to the beads were detected using immunoblotting. (E) Quantification of proteins based on immunoblots from three independent experiments. Levels of proteins were normalized to those of AP2 α. Error bars indicate SD. (F) Representative SIM images showing the subcellular localization of clathrin heavy chain (CHC), AP2 α, and CCDC32, focusing on the plasma membrane. Endogenous CHC was stained using anti-CHC and Alexa Fluor 647–conjugated secondary antibodies, whereas endogenous AP2 α was stained using anti-α antibodies and Alexa Fluor 568–conjugated secondary antibodies. FLAG-tagged CCDC32 was labeled using Alexa Fluor 488–conjugated anti-FLAG antibodies. Colocalization of CHC and AP2 α, but not with CCDC32, was indicated by arrows. (Scale bars, 10 μm for main images and 1 μm for enlarged images.) (G) Profile analysis plot comparing the distributions of CHC, α and CCDC32 within the rectangular areas of F. (H) Quantification of the colocalization of CCDC32 with AP2 adaptor (α staining) and CHC using the Pearson correlation coefficient. Images were captured as in F and analyzed using ImageJ. Each dot represents imaging data of an individual cell focusing on the plasma membrane. In randomized data, CHC and AP2 α images were rotated 90 degrees clockwise, whereas CCDC32 images were not rotated. Error bars indicate SD. n.s., P > 0.05 (calculated using Student’s t test).

Article Snippet: The 3xFLAG- AAGAB protein was transiently expressed in HEK 293T cells and immunoprecipitated using anti- FLAG M2 antibodies and Protein A/G agarose beads (Santa Cruz Biotechnology, #sc- 2003).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Plasmid Preparation, Control, Immunoprecipitation, Isolation, Incubation, Clinical Proteomics, Membrane, Staining, Labeling, Imaging