v5 tag  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: The protein phosphatase 2A-B56α complex regulates N-Myc degradation in neuroblastoma

doi: 10.1016/j.jbc.2026.111298

Figure Lengend Snippet: PP2A activity is dependent on the N-Myc phosphorylation sites S62 and T58. A , Kelly cells were transduced with pLenti6.3 expressing V5-tagged WT N-Myc and mutant S62D or T58A N-Myc. Cells were treated with 25 μg/ml cycloheximide in a chase experiment to assess protein stability over 60 min by immunoblot of whole cell lysate for V5-tagged N-Myc. B , quantification of V5-tagged N-Myc plotted after normalization to Vinculin and the untreated control. Data plotted as mean ± S.D. are from four independent experiments ( n = 4), and half-lives were determined from exponential decay curve fitting. C , Kelly cells expressing the WT, S62D, and T58A N-Myc are treated with DMSO or 20 μM DT-061 for 3 h and immunoblotted for V5-tagged N-Myc, representative images are presented. D , quantification of V5-tagged N-Myc after normalization to Vinculin and DMSO controls from three independent experiments ( n = 3). Statistical significance was determined by two-way ANOVA; ∗ p < 0.05; ns, not significant. E , representative images of clonogenic assays of Kelly WT and S62D N-Myc plated at low density and treated with DT-061 for 14 days. F , colony formation assays from panel E were quantified by absorbance of dissolved crystal violet stained colonies, and plotted data represent the mean ± S.D. of three independent biological replicates (two technical replicates for each biological replicate).

Article Snippet: Membranes were blocked in 5% non-fat milk/TBST for 1 hour and incubated overnight at 4 °C with primary antibodies: c-Myc (ABclonal, A19032, 1:1000), phospho S62 c-Myc (Abcam, ab185656, 1:1000), N-Myc (Abcam, ab16898, 1:1000), Vinculin (Santa Cruz Biotech, sc-73614, 1:5,000), GAPDH (Santa Cruz Biotech, sc-32233, 1:2000), V5-tag (Cell Signaling Technology, 13,202, 1:1000), AP2β (Cell Signaling Technology, 2509, 1:1000), GATA3 (Cell Signaling Technology, 5852, 1:1,000), PHOX2B (Santa Cruz Biotech, sc-376997, 1:1,000), PARP (Cell Signaling Technology, 9542L, 1:1000), Cleaved caspase-3 (Cell Signaling Technology, 9664L, 1:1,000), and PPP2R2A (Santa Cruz Biotech, sc-81606, 1:1000).

Techniques: Activity Assay, Phospho-proteomics, Transduction, Expressing, Mutagenesis, Western Blot, Control, Staining

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

Article Title: HPV16 E6 oncoprotein promotes microhomology-mediated viral integration by increasing PolΘ protein expression

doi: 10.1073/pnas.2532479123

Figure Lengend Snippet: HPV16 E6 enhances MMEJ pathway activity and MMEJ-mediated DNA integration efficiency. ( A ) Immunoblot of HA and α-tubulin in U2OS cells expressing empty vector (EV), HA-tagged HPV16 E6, or E7. ( B ) Relative MMEJ activity measured using the I-SceI-based MMEJ reporter assay in U2OS cells expressing EV, HPV16 E6, or E7. Data are shown as the mean ± SD. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test (** P < 0.01; **** P < 0.0001). ( C ) Relative MMEJ activity measured using the CRISPR-based MMEJ reporter assay in HPV-positive UPCI-SCC-154 cells with or without HPV16 E6 knockout. Data are shown as the mean ± SD. Statistical significance was calculated using two-tailed Student’s t test (**** P < 0.0001). ( D ) Relative MMEJ activity measured using the CRISPR-based MMEJ reporter assay in HPV-positive UPCI-SCC-154 cells with HPV16 E6 & E7 double knockout and complementation as indicated. Data are shown as mean ± SD. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test (**** P < 0.0001; ns, not significant). ( E ) Immunoblot of V5-tagged endogenous PolΘ, other key MMEJ pathway components (PARP1, LIG3, XRCC1, APEX2, HMCES, FEN1), and GAPDH in V5 - POLQ knock-in U2OS cells expressing EV, HPV16 E6, or E7. ( F ) Immunoblot of endogenous PolΘ, other MMEJ pathway components, and GAPDH in HPV-positive UPCI-SCC-154 cells with or without HPV16 E6 knockout. ( G ) Immunofluorescence analysis of V5-POLQ knock-in U2OS cells expressing EV or HPV16 E6. Left : representative images showing V5 (endogenous PolΘ), DAPI, and merged channels. (Scale bar, 2 μm.) Right : quantification of V5-PolΘ foci per cell. At least 150 cells were analyzed per condition. Data are shown as mean ± SD. Statistical significance was calculated using two-tailed Student’s t test (**** P < 0.0001). ( H ) Schematic representation of the reporter system designed to measure MMEJ-mediated double-strand DNA integration efficiency [Created in BioRender. Zhu, G. (2026) https://BioRender.com/deojl1w ]. ( I ) Relative MMEJ-mediated integration efficiency in HEK293T cells expressing HPV16 E6 or control vector. Controls included cells transfected with donor DNA alone, donor DNA plus nontargeting sgRNA (sg Luc ), or donor DNA plus both sg Luc and sg FBL . Data are shown as the mean ± SD. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test (**** P < 0.0001).

Article Snippet: The slides were incubated with anti-V5 antibodies (Cell Signaling Technology, #13202S) at 4 ∘ C overnight, followed by incubation with secondary antibodies (Invitrogen, #A-11008) for 1 h at room temperature.

Techniques: Activity Assay, Western Blot, Expressing, Plasmid Preparation, Reporter Assay, CRISPR, Knock-Out, Two Tailed Test, Double Knockout, Knock-In, Immunofluorescence, Control, Transfection

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

Article Title: HPV16 E6 oncoprotein promotes microhomology-mediated viral integration by increasing PolΘ protein expression

doi: 10.1073/pnas.2532479123

Figure Lengend Snippet: HPV16 E6 promotes MMEJ repair through a UBE3A-dependent and p53-independent mechanism. ( A ) Immunoblot of UBE3A, p53, HA-tagged HPV16 E6, and GAPDH proteins in WT, TP53 -knockout, or UBE3A -knockout U2OS cells expressing EV or WT HPV16 E6. ( B ) Relative MMEJ activity measured using the I-SceI-based MMEJ GFP reporter assay in WT, TP53 -knockout, or UBE3A -knockout U2OS cells expressing EV or WT HPV16 E6. ( C ) Immunofluorescence analysis of WT or UBE3A -knockout, V5-POLQ knock-in U2OS cells with or without HPV16 E6 expression. Left : representative images showing V5 (endogenous PolΘ), DAPI, and merged channels. (Scale bar, 2 μm.) Right : quantification of V5-PolΘ foci per cell. At least 150 cells were analyzed per condition. Data are shown as mean ± SD. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test (**** P < 0.0001; ns, not significant). ( D ) Relative MMEJ activity evaluated using the CRISPR -based reporter assay in WT, TP53 -knockout, or UBE3A -knockout, HPV-positive UPCI-SCC-154 cells with or without HPV16 E6 knockout. ( E ) Immunoblot of p53, HA-tagged WT or mutant HPV16 E6, and GAPDH proteins in MMEJ reporter U2OS cells expressing the indicated E6 constructs. ( F ) Relative MMEJ activity measured using the I-SceI-based MMEJ GFP reporter assay in U2OS cells expressing EV, WT, or mutant HPV16 E6. ( G ) Immunoblot of FLAG-tagged WT UBE3A or catalytically inactive UBE3A C820A mutant, p53, HA-tagged E6, and GAPDH in U2OS cells expressing EV, WT, or UBE3A C820A mutant, with or without HPV16 E6 expression. ( H ) Relative MMEJ activity measured using the I-SceI-based MMEJ GFP reporter assay in U2OS cells expressing EV, WT UBE3A, or UBE3A C820A mutant, with or without HPV16 E6 expression.

Article Snippet: The slides were incubated with anti-V5 antibodies (Cell Signaling Technology, #13202S) at 4 ∘ C overnight, followed by incubation with secondary antibodies (Invitrogen, #A-11008) for 1 h at room temperature.

Techniques: Western Blot, Knock-Out, Expressing, Activity Assay, Reporter Assay, Immunofluorescence, Knock-In, CRISPR, Mutagenesis, Construct

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

Article Title: HPV16 E6 oncoprotein promotes microhomology-mediated viral integration by increasing PolΘ protein expression

doi: 10.1073/pnas.2532479123

Figure Lengend Snippet: RAD23A mediates the impact of E6 on MMEJ activity by regulating PolΘ stability. ( A ) Co-IP analysis. Whole cell lysates from UBE3A -KO HEK293T cells transduced with indicated constructs were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting of input and IP samples for FLAG (UBE3A), HA-tagged HPV16 E6, p53, RAD23A, and GAPDH. ( B ) Relative MMEJ activity measured using the I-SceI-based MMEJ GFP reporter assay in WT, E6 expressing, or RAD23A -knockout U2OS cells. Data are shown as the mean ± SD. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test (** P < 0.01; *** P < 0.001; ns, not significant). ( C ) Immunoblot of endogenous PolΘ, RAD23A, and GAPDH in U2OS cells with or without RAD23A knockout. ( D ) Relative MMEJ activity measured using the I-SceI-based MMEJ GFP reporter assay in U2OS cells overexpressing EV, WT RAD23A, or RAD23A ΔUBL mutant. Data are shown as the mean ± SD. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test (** P < 0.01; **** P < 0.0001). ( E ) Immunoblot of endogenous PolΘ, V5 (RAD23A), RAD23A, and GAPDH in U2OS cells overexpressing EV, WT RAD23A, or RAD23A ΔUBL mutant. ( F ) Immunoblot of endogenous PolΘ, V5 (RAD23A), RAD23A, HA (ubiquitin), and GAPDH in HEK293T cells overexpressing EV or ubiquitin together with WT RAD23A or RAD23A ΔUBL mutant.

Article Snippet: The slides were incubated with anti-V5 antibodies (Cell Signaling Technology, #13202S) at 4 ∘ C overnight, followed by incubation with secondary antibodies (Invitrogen, #A-11008) for 1 h at room temperature.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Transduction, Construct, Immunoprecipitation, Western Blot, Reporter Assay, Expressing, Knock-Out, Mutagenesis, Ubiquitin Proteomics

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

Article Title: HPV16 E6 oncoprotein promotes microhomology-mediated viral integration by increasing PolΘ protein expression

doi: 10.1073/pnas.2532479123

Figure Lengend Snippet: RAD23A interacts with PolΘ and promotes its ubiquitination. ( A ) Co-IP analysis. Whole cell lysates from POLQ -knockout HEK293T cells reconstituted with EV or HA - Ub , together with 3× FLAG -tagged full-length POLQ , with or without MG132 treatment, were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting of input and IP samples for FLAG (PolΘ) and HA (Ubiquitin). ( B ) Co-IP analysis. Whole cell lysates from POLQ -knockout HEK293T cells reconstituted with EV or 3× FLAG -tagged full-length POLQ , together with V5-RAD23A, with or without MG132 treatment, were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting of input and IP samples for FLAG (PolΘ) and V5 (RAD23A). ( C ) Co-IP analysis. Whole cell lysates from POLQ -knockout HEK293T cells reconstituted with EV, 3× FLAG -tagged full-length POLQ or its truncated mutants (1-950, 1-1750, 1751-2590), together with V5-RAD23A, with or without HA-ubiquitin, were immunoprecipitated with anti-V5 antibody, followed by immunoblotting of input and IP samples for FLAG (PolΘ fragments) and RAD23A. ( D ) Schematic of the working model for HPV16 E6-enhanced MMEJ-mediated viral integration [Created in BioRender. Zhu, G. (2026) https://BioRender.com/3g525ja ].

Article Snippet: The slides were incubated with anti-V5 antibodies (Cell Signaling Technology, #13202S) at 4 ∘ C overnight, followed by incubation with secondary antibodies (Invitrogen, #A-11008) for 1 h at room temperature.

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Knock-Out, Immunoprecipitation, Western Blot