Journal: Journal of Cell Science

Article Title: UBQLN2 is necessary for UBE3A-mediated proteasomal degradation of the domesticated retroelement PEG10

doi: 10.1242/jcs.264105

Figure Lengend Snippet: The UBE3A AZUL domain is important for interaction with UBQLN2. (A) WT HEK293 cell lysate was subjected to an IP with anti-UBQLN2 antibody or IgG control and detected by western blotting. UBQLN1/2 were detected using an antibody that recognizes both ubiquilins but was precipitated with an antibody that only recognizes UBQLN2. n =2. (B) WT HEK293 were transfected with FLAG-tagged UBE3A constructs. After 48 h, cells were lysed and UBQLN2 was immunoprecipitated by anti-UBQLN2 antibody or IgG control, followed by western blotting. UBE3A was detected with FLAG antibody and PEG10 was detected with a polyclonal antibody. n =3. (C) UBE3A binds to UBQLN2 in a UBA-independent manner. TKO HEK293 cells were co-transfected with the FLAG-tagged UBE3A and FLAG-UBQLN2 constructs shown. UBQLN2 was immunoprecipitated using a UBQLN2 antibody and probed against UBE3A with a UBE3A antibody. n =2. IB, immunoblot.

Article Snippet: Primary antibodies for PEG10 (Proteintech 14412-1-AP; 1:1000), HA-tag (either Proteintech 66006-2-Ig or Cell Signaling Technology C29F4; 1:5000), UBQLN2/1 (5F5 clone, Novus Biologicals; 1:1000), UBQLN2 (6H9 clone, Novus Biologicals; 1:1000), M2 FLAG-tag (Sigma-Aldrich F3165; 1:5000), UBE3A (Proteintech 10344-1-AP; 1:1000), PSMD4 (Cell Signaling Technology 12441S; 1:1000), MYC tag (Cell Signaling Technology 2276S; 1:2000), acetylated lysine (CST Ac-K-100 6952S; 1:1000), poly/mono-ADP Ribose (D9P7Z clone, Cell Signaling Technology; 1:1000) and tubulin (DM1A clone, Novus Biologicals; 1:10,000) were left on overnight at 4°C, while secondary antibodies (IR680 and IR800 conjugates, LI-COR) were incubated for 1 h at room temperature at 1:10,000 concentration.

Techniques: Control, Western Blot, Transfection, Construct, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: UBQLN2 is necessary for UBE3A-mediated proteasomal degradation of the domesticated retroelement PEG10

doi: 10.1242/jcs.264105

Figure Lengend Snippet: UBE3A and UBQLN2 are both required to control levels of gag-pol protein. (A) Representative western blot of UBE3A knockdown in WT and TKO HEK293 cells. Cells were transfected with 100 pmol siRNA against a non-targeting construct (NTC) or UBE3A and harvested for western blotting 72 h later. PEG10 and UBE3A were visualized with polyclonal antibodies. Shown is one of three representative experiments. (B) Quantification of UBE3A protein abundance from the experiment shown in A. (C) Quantification of gag protein abundance from the experiment shown in A. (D) Gag-pol protein abundance from the experiment shown in A. For B–D, results for each band were normalized to a blot average of that band for each independent experiment, and statistics were determined by two-way ANOVA with Šídák's multiple comparisons. Shown is mean±s.e.m., n =3. (E) Western blot of CRISPR/Cas9 UBE3A knockout cell lines. TKO HEK293 cells were used as a genetic background, so no ubiquilin expression is evident. (F) Rescue experiment of TKO/UBE3A knockout cell line with transfection of UBE3A and UBQLN2 constructs. Delta symbol signifies transfection of UBE3A ΔAZUL . Cells were transfected for 48 h and lysed for western blotting using polyclonal PEG10 and UBE3A antibodies, and a UBQLN2 antibody that recognizes both UBQLN1 and UBQLN2. Shown is one of three representative experiments using clone #48. (G) Quantification of gag-pol protein levels from the experiment shown in F. Delta symbol signifies transfection of UBE3A ΔAZUL . Gag-pol levels were quantified, normalized to tubulin for each well, and then normalized to a blot average. Shown is mean±s.e.m. for three independent experiments. Statistics were determined by two-way ANOVA with Šídák's multiple comparisons. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. IB, immunoblot.

Article Snippet: Primary antibodies for PEG10 (Proteintech 14412-1-AP; 1:1000), HA-tag (either Proteintech 66006-2-Ig or Cell Signaling Technology C29F4; 1:5000), UBQLN2/1 (5F5 clone, Novus Biologicals; 1:1000), UBQLN2 (6H9 clone, Novus Biologicals; 1:1000), M2 FLAG-tag (Sigma-Aldrich F3165; 1:5000), UBE3A (Proteintech 10344-1-AP; 1:1000), PSMD4 (Cell Signaling Technology 12441S; 1:1000), MYC tag (Cell Signaling Technology 2276S; 1:2000), acetylated lysine (CST Ac-K-100 6952S; 1:1000), poly/mono-ADP Ribose (D9P7Z clone, Cell Signaling Technology; 1:1000) and tubulin (DM1A clone, Novus Biologicals; 1:10,000) were left on overnight at 4°C, while secondary antibodies (IR680 and IR800 conjugates, LI-COR) were incubated for 1 h at room temperature at 1:10,000 concentration.

Techniques: Control, Western Blot, Knockdown, Transfection, Construct, Quantitative Proteomics, CRISPR, Knock-Out, Expressing

Journal: bioRxiv

Article Title: Whole-Proteome Screening and Multi-Modal Profiling of Antigen-Specific CD4+ T Cells at Single-Cell Resolution

doi: 10.1101/2025.04.21.649828

Figure Lengend Snippet: a. HPV-16 therapeutic vaccine clinical trial design. Patients received vaccines targeting E6 and E7 proteins at Week 0, 4, and 8. PBMCs were collected pre-vaccination, at Week 10, and at Week 15 post-series completion. b. left, UMAP clustering of CD4+ T cells from PBMCs of HPV16+ patients with persistent precancerous lesions. right, HPV16-specific CD4+ T cells identified by SCT-dextramers. c. Highlighting of H1-H5 TCR clones among HPV16-specific CD4+ T cells. d. Expression of signature gene markers for defining phenotype clusters. Mean expression levels were normalized across groups. e. Engineering of primary CD4+ T cells with HPV-specific TCRs. CD4+ T cells were enriched from haplotype-matched PBMCs, endogenous TCRs were knocked out using CRISPR, and HPV TCRs were transduced via lentivirus. f. Validation of TCR specificity by tetramer binding (n=3) . g. Transduction efficiency of HPV-specific CD4 TCRs into primary CD4+ versus CD8+ T cells. h. Alanine scanning to identify key TCR recognition motifs. H2- and H5-TCR-transduced CD4+ T cells were cocultured with DR1+ K562 cells pulsed with alanine-substituted 9-mer core peptides (A1-A9) across 0.001 – 1 μM. IFNγ secretion was measured (OD 450 ). i. Cross-reactivity analysis against human self-antigens. H2- and H5-TCR-transduced T cells were co-cultured with LCLs pulsed with peptides identified from BLAST search and motif scans. H2 TCR used a single 1 µM dose; H5 TCR was dose titrated at 1, 2, and 10 µM. j. Alloreactivity screen. H2- and H5-TCR-transduced CD4+ T cells were tested against 41 LCL lines representing 92 distinct class II HLA alleles. DRB1*01:01-positive LCLs ( n=4 ) pulsed with E6 91-107 or E6 129-142 served as positive controls. Alloreactivity of H5 to HLA-DRB1*13:05-positive LCLs is marked with orange arrows. k. Confirmation of H2 TCR reactivity to naturally processed E6 antigen. DRB1*01:01+ LCL (GM17281B) was titrated with full-length E6 protein (0-10 μg/mL), co-cultured with H2-TCR-transduced CD4+ T cells, and evaluated for IFNγ secretion. A DRB1*01:01-LCL (GM12244A) served as a negative control.

Article Snippet: A DRB1*01:01+ LCL (GM17281B) was pulsed with E6 protein (Rndsystems, AP-120) at 10 μg/mL, 2 μg/mL, 1 μg/mL, and 0 μg/mL for 3 hours, and then co-cultured with H2-TCR-transduced CD4+ T cells overnight.

Techniques: Vaccines, Clone Assay, Expressing, CRISPR, Binding Assay, Transduction, Cell Culture, Negative Control

Journal: Journal of Immunotherapy (Hagerstown, Md. : 1997)

Article Title: A Novel Cell-based Luciferase Reporter Platform for the Development and Characterization of T-Cell Redirecting Therapies and Vaccine Development

doi: 10.1097/CJI.0000000000000453

Figure Lengend Snippet: TCR-KO cells as a platform for sensitive detection of vaccine-induced TCR activation. A, HA1.7 TCR reporter cells were activated with HA peptides, as indicated, in the presence of A375 target cells. B, HA1.7 TCR reporter cells were activated with HA peptide in the presence of THP-1 or THP-1-DC cells. C, HA1.7 TCR reporter cells were activated with recombinant H3-loaded THP-1-DCs. D, HA1.7 TCR reporter cells were activated with Fluzone-loaded THP-1-DCs. E, HPV16 E6 TCR reporter cells were activated with A375 cells that were transiently transfected with a titration of HPV16 E6 expression plasmid. Data is representative of ≥2 independent experiments. HA indicates hemagglutinin; HPV, human papillomavirus; KO, knockout; TCR, T-cell receptor; THP-1-DC, THP-1-derived dendritic cell.

Article Snippet: HPV16 E6 expression plasmid was from OriGene.

Techniques: Activation Assay, Recombinant, Transfection, Titration, Expressing, Plasmid Preparation, Knock-Out, Derivative Assay

Journal: Journal of Virology

Article Title: PDZRN3/LNX3 Is a Novel Target of Human Papillomavirus Type 16 (HPV-16) and HPV-18 E6

doi: 10.1128/JVI.01743-14

Figure Lengend Snippet: (A) HPV-16 and HPV-18 E6 proteins induce degradation of PDZRN3. Plasmids expressing FLAG-tagged PDZRN3A and HA-tagged PDZRN3B were transfected into 293 cells alone or with plasmids expressing HPV-16 or HPV-18 E6. Cell extracts were analyzed by Western blotting with anti-HA and anti-FLAG antibodies; β-galactosidase was included as a control for transfection efficiency. (B) HPV-18 E6-induced degradation of PDZRN3 is PDZ binding dependent. (Top) Plasmids expressing FLAG-tagged PDZRN3A and HA-tagged PDZRN3B were transfected into 293 cells alone or with plasmids expressing wild-type HPV-18 E6 or the PDZ binding-defective HPV-18 E6T156E mutant. Cell extracts were analyzed by Western blotting with anti-HA and anti-FLAG antibodies; β-galactosidase was included as a control for transfection efficiency. (Bottom) Extracts of 293 cells transfected with pCDNA3.1 (lanes C) or pCDNA-FLAG-PDZRN3A (lanes P) were incubated with GST, GST-HPV18E6, or GST-HPV18E6T156E, as indicated. After washing, the bound proteins were analyzed by a Western blot assay probed with anti-PDZRN3 and anti-GST antibodies to confirm equal loading of GST proteins. (C) HPV-18 E6-induced degradation of PDZRN3 is proteasome dependent. A plasmid expressing FLAG-tagged PDZRN3A was transfected into 293 cells alone or with an HPV-18 E6-expressing plasmid. After overnight incubation, the cells were treated with the proteasome inhibitors carbobenzoxy-Leu-Leu-leucinal (CBZ) and N-acetyl-l-leucyl-l-leucyl-l-norleucinal for 3 h before harvesting. Cell extracts were analyzed by Western blotting with anti-FLAG antibody; β-galactosidase was included as a control for transfection efficiency.

Article Snippet: A degradation assay ( ) with the radiolabeled, in vitro -translated PDZRN3 and HPV E6 proteins (TNT kit; Promega), showing that HPV-16 and HPV-18 E6 induces degradation of PDZRN3ΔRING in vitro , confirms that the interaction is robust. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 2 caption a7 The RING domain of PDZRN3 is not required for E6-induced degradation. (A) Plasmids expressing PDZRN3A and PDZRN3AΔRING were transfected into 293 cells alone or with plasmids expressing HPV-16 or HPV-18 E6.

Techniques: Expressing, Transfection, Western Blot, Binding Assay, Mutagenesis, Incubation, Plasmid Preparation

Journal: Journal of Virology

Article Title: PDZRN3/LNX3 Is a Novel Target of Human Papillomavirus Type 16 (HPV-16) and HPV-18 E6

doi: 10.1128/JVI.01743-14

Figure Lengend Snippet: The RING domain of PDZRN3 is not required for E6-induced degradation. (A) Plasmids expressing PDZRN3A and PDZRN3AΔRING were transfected into 293 cells alone or with plasmids expressing HPV-16 or HPV-18 E6. Cell extracts were analyzed by Western blotting with an anti-PDZRN3 antibody (upper panel); β-galactosidase was included as a control for transfection efficiency (lower panel). (B) In vitro-translated radiolabeled PDZRN3A and PDZRN3AΔRING (arrow) and HPV-16 or HPV-18 E6 were incubated together at 30°C for 1 and 2 h, as indicated, and then analyzed by SDS-PAGE and autoradiography; positions of molecular weight markers are shown on the right. The inputs are shown in the lower panels. (C) Knockdown of HPV-18 E6 in HeLa cells results in increased levels of endogenous PDZRN3. HeLa cells were transfected with siRNA to luciferase (Luci), HPV-18 E6/E7, PDZRN3, or p53 (Dharmacon), and total cell extracts were harvested at 48 h posttransfection. Cell extracts were analyzed by Western blotting for the proteins indicated; α-tubulin was included as a loading control. The histogram shows the collated results of five experiments quantitated with ImageJ software and subjected to statistical analysis by the Prism program.

Article Snippet: A degradation assay ( ) with the radiolabeled, in vitro -translated PDZRN3 and HPV E6 proteins (TNT kit; Promega), showing that HPV-16 and HPV-18 E6 induces degradation of PDZRN3ΔRING in vitro , confirms that the interaction is robust. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 2 caption a7 The RING domain of PDZRN3 is not required for E6-induced degradation. (A) Plasmids expressing PDZRN3A and PDZRN3AΔRING were transfected into 293 cells alone or with plasmids expressing HPV-16 or HPV-18 E6.

Techniques: Expressing, Transfection, Western Blot, In Vitro, Incubation, SDS Page, Autoradiography, Molecular Weight, Luciferase, Software

Journal: Journal of Virology

Article Title: PDZRN3/LNX3 Is a Novel Target of Human Papillomavirus Type 16 (HPV-16) and HPV-18 E6

doi: 10.1128/JVI.01743-14

Figure Lengend Snippet: (A) Knockdown of HPV-18 E6 in HeLa cells increases nuclear PDZRN3 levels. HeLa cells were seeded onto coverslips and transfected with siRNA for luciferase or HPV-18 E6/E7 (Dharmacon). After 48 h, the cells were fixed, permeabilized, and then probed for p53 (red) and PDZRN3 (green). Each panel shows two different fields of view. (B) In C33A cells, transfection of HPV-18 E6 reduces nuclear PDZRN3 in a PBM-dependent manner. C33A cells were seeded onto coverslips and transfected with pCDNA3.1, pCDNA3:HA-HPV-18E6, or pCDNA3:HA-HPV18E6T156E. After 24 h, the cells were fixed, permeabilized, and then probed for HA (red) and PDZRN3 (green). Arrows indicate cells expressing E6 with a corresponding reduction in nuclear PDZRN3 with wild-type E6 but no change with the T156E mutant.

Article Snippet: A degradation assay ( ) with the radiolabeled, in vitro -translated PDZRN3 and HPV E6 proteins (TNT kit; Promega), showing that HPV-16 and HPV-18 E6 induces degradation of PDZRN3ΔRING in vitro , confirms that the interaction is robust. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 2 caption a7 The RING domain of PDZRN3 is not required for E6-induced degradation. (A) Plasmids expressing PDZRN3A and PDZRN3AΔRING were transfected into 293 cells alone or with plasmids expressing HPV-16 or HPV-18 E6.

Techniques: Transfection, Luciferase, Expressing, Mutagenesis

Journal: Journal of Virology

Article Title: PDZRN3/LNX3 Is a Novel Target of Human Papillomavirus Type 16 (HPV-16) and HPV-18 E6

doi: 10.1128/JVI.01743-14

Figure Lengend Snippet: (A) Knockdown of HPV-18 E6 in HeLa cells decreases levels of phosphorylated STAT5β. HeLa cells were transfected with siRNA to luciferase, PDZRN3, HPV18 E6, or E6AP, and total cell extracts were harvested at 72 h posttransfection. (Left panel) IFN-α treatment prior to harvesting specifically induces phosphorylation of STAT5β. (Right panel) Cell extracts were analyzed by Western blotting for the proteins indicated; γ-tubulin was included as a control. The histogram shows the collated results of at least three experiments quantitated with ImageJ software and subjected to statistical analysis by the Prism program. (B) Transfection of PDZRN3 in 293 cells reduces levels of phosphorylated endogenous STAT5β, which is rescued by cotransfection of HPV-18 E6. HEK293 cells were transfected with plasmids expressing PDZRN3 with or without plasmids expressing HPV-18 E6. Empty plasmid was transfected as a control. The cells were extracted in the presence of phosphatase inhibitors, and the extracts were analyzed by SDS-PAGE and Western blotting for the proteins indicated. The histogram shows the collated results of at least three experiments quantitated with ImageJ software and subjected to statistical analysis by the Prism program. (C) HPV-31 E6 binds to PDZRN3 more weakly than HPV-16 or HPV-18 E6. Extracts from 293 cells transfected with pCDNA3.1 (−) or pCDNA-FLAG-PDZRN3A (+) were incubated with GST-HPV31E6, GST-HPV16E6, GST-HPV18E6, or GST-HPV18E6T156E as indicated. After washing, the bound proteins were analyzed by a Western blot assay probed with anti-PDZRN3 and anti-GST antibodies to confirm equal loading of GST proteins. The blot was reprobed for DLG1 to confirm GST-E6 integrity. (D) HPV-31 E6 is less efficient than HPV-16 E6 at inducing PDZRN3 degradation in vitro. In vitro-translated PDZRN3 was incubated at 30°C either alone or with in vitro-translated HPV-16 or HPV-31 E6 as indicated. The remaining PDZRN3 was analyzed by SDS-PAGE and autoradiography. Dlg1 and p53 were included as controls. The values below the lanes are the percentages of the input protein remaining and are the mean results of two assays.

Article Snippet: A degradation assay ( ) with the radiolabeled, in vitro -translated PDZRN3 and HPV E6 proteins (TNT kit; Promega), showing that HPV-16 and HPV-18 E6 induces degradation of PDZRN3ΔRING in vitro , confirms that the interaction is robust. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG 2 caption a7 The RING domain of PDZRN3 is not required for E6-induced degradation. (A) Plasmids expressing PDZRN3A and PDZRN3AΔRING were transfected into 293 cells alone or with plasmids expressing HPV-16 or HPV-18 E6.

Techniques: Transfection, Luciferase, Western Blot, Software, Cotransfection, Expressing, Plasmid Preparation, SDS Page, Incubation, In Vitro, Autoradiography