Journal: bioRxiv

Article Title: Intrinsic immunity against HAdV is achieved by a novel epigenetic silencing complex

doi: 10.1101/2025.02.10.637372

Figure Lengend Snippet: (A) H1299 cells were infected with HAdV-wt at a MOI of 50 and harvested 24 h, 48 h and 72 h pi. Whole cell lysates were prepared, separated by SDS PAGE, and subjected to immunoblotting using mAb AC-15 (β-actin), rabbit pAb MPP8, rabbit pAb PPHLN1, rabbit pAb TASOR, rabbit pAb NP220, mouse mAb E2A, and mouse mAb E1B-55K. β-actin serves as a loading control. Molecular weights in kDa are indicated in the left, proteins are indicated on the right. (B) H1299 cells were infected with HAdV-wt and mutant versions depleted for early viral proteins, ΔE1B- 55K, ΔE4orf6, and ΔE4orf3, at a MOI of 50. In addition, we transiently transfected two samples with p53 DNA as indicated. Cells were harvested 48 h pi and whole cell lysates were prepared. Proteins were separated by SDS PAGE and subjected to immunoblotting using mAb AC-15 (β-actin), mouse mAb MPP8, rabbit pAb PPHLN1, rabbit pAb TASOR, mouse mAb E2A, mouse mAb E1B-55K, mouse mAb E4orf6, and rat mAb E4orf3. β-actin serves as a loading control. Molecular weights in kDa are indicated in the left, proteins are indicated on the right. (C) Illustration of the viral E3 ubiquitin ligase targeting p53 for proteasomal degradation during HAdV infection. Created with Biorender.com .

Article Snippet: Primary antibodies used for immunoblotting are mouse mAb AC-15 (anti-β-actin; Sigma-Aldrich), mouse mAb MPP8 (sc-398598; Santa Cruz Biotechnology), rabbit pAb MPP8 (HPA040035; Sigma-Aldrich), rabbit pAb PPHLN1 (HPA0038902; Sigma-Aldrich), rabbit pAb TASOR (FAM208A; NBP1-90673; Novus Biologicals), rabbit pAb NP220 (A301-547A; Biomol) mouse mAb Ubiquitin (P4D1; Cell Signaling), rabbit pAb PML (ab72137, abcam), mouse mAb FLAG(M2) (F1804, Sigma Aldrich), mouse mAb E1A (M73; sc-25, Santa Cruz Biotechnology; [ ]), mouse mAb E1B-55K (4E8, [ ]), mouse mAb E2A (B6-8, [ ]), rat mAb E4orf3 (6A11, [ ]), mouse mAb E4orf6 (RSA3, [ ]), and rabbit pAb Capsid (L133, [ ]).

Techniques: Infection, SDS Page, Western Blot, Control, Mutagenesis, Transfection

Journal: bioRxiv

Article Title: Intrinsic immunity against HAdV is achieved by a novel epigenetic silencing complex

doi: 10.1101/2025.02.10.637372

Figure Lengend Snippet: (A) H1299 cells were infected with HAdV-wt at a MOI of 50 and treated with proteasome inhibitor MG132. Cells were harvested 48 h pi and whole cell lysates were prepared. Proteins were separated by SDS PAGE and subjected to immunoblotting using mAb AC-15 (β-actin), mouse mAb MPP8, rabbit pAb PPHLN1, rabbit pAb TASOR, mouse mAb Ubiquitin and mouse mAb E2A. β-actin serves as a loading control. Molecular weights in kDa are indicated in the left, proteins are indicated on the right. (B) Graphical representation of the viral E3 ubiquitin ligase composed of early viral proteins E1B-55K and E4orf6. MPP8 is targeted by E1B-55K and E4orf6 for proteasomal degradation, which can be inhibited by MG132 treatment. (C, D) H1299 cells were infected with HAdV-wt at a MOI of 50 and harvested 48 h pi. Co-immunoprecipitation assay was performed by using a (C) mouse mAb MPP8 or (D) rabbit pAb TASOR. Proteins were separated by SDS PAGE and subjected to immunoblotting using mAb AC-15 (β-actin), mouse mAb E1B-55K, (C) mouse mAb MPP8 or (D) rabbit pAb TASOR. β-actin serves as a loading control. Molecular weights in kDa are indicated in the left, proteins are indicated on the right.

Article Snippet: Primary antibodies used for immunoblotting are mouse mAb AC-15 (anti-β-actin; Sigma-Aldrich), mouse mAb MPP8 (sc-398598; Santa Cruz Biotechnology), rabbit pAb MPP8 (HPA040035; Sigma-Aldrich), rabbit pAb PPHLN1 (HPA0038902; Sigma-Aldrich), rabbit pAb TASOR (FAM208A; NBP1-90673; Novus Biologicals), rabbit pAb NP220 (A301-547A; Biomol) mouse mAb Ubiquitin (P4D1; Cell Signaling), rabbit pAb PML (ab72137, abcam), mouse mAb FLAG(M2) (F1804, Sigma Aldrich), mouse mAb E1A (M73; sc-25, Santa Cruz Biotechnology; [ ]), mouse mAb E1B-55K (4E8, [ ]), mouse mAb E2A (B6-8, [ ]), rat mAb E4orf3 (6A11, [ ]), mouse mAb E4orf6 (RSA3, [ ]), and rabbit pAb Capsid (L133, [ ]).

Techniques: Infection, SDS Page, Western Blot, Control, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Intrinsic immunity against HAdV is achieved by a novel epigenetic silencing complex

doi: 10.1101/2025.02.10.637372

Figure Lengend Snippet: H1299 shCRL, shMPP8, sh PPHLN1, and shTASOR were infected with HAdV-wt at a MOI of 50 and harvested 48 h pi. (A) Viral particles were isolated from HAdV-infected cells by three cycles of freeze and thaw. HEK293 cells were reinfected with a serial dilution of the viral particles and virus yield was determined using quantitative E2A immunofluorescence staining. Bar charts represents mean values of three independent biological replicates. H1299 shCRL cells were set to 100% and the remaining samples were normalized. Statistically significant differences were determined using an one-way ANOVA and Dunnet’s T3 test with the GraphPad Prism10 software. ns = not significant; * p ≤ 0.05. (B) Whole cell lysates were prepared from some of the cells, from which virus particles were isolated. Proteins were separated by SDS PAGE and subjected to immunoblotting using mAb AC-15 (β-actin), rabbit pAb MPP8, rabbit pAb PPHLN1, rabbit pAb TASOR, and mouse mAb E2A. β-actin serves as a loading control. Molecular weights in kDa are indicated in the left, proteins are indicated on the right. (C,D) Total mRNA was isolated using TRIzol reagent, reverse transcribed and subjected to qPCR using oligonucleotides specific for viral transcripts (C) E1A and (D) Hexon. The data was normalized on the respective GAPDH, actin and RPL30 mRNA levels. Bar charts represents mean values of three independent biological replicates measured in technical triplicates. Statistically significant differences were determined using an unpaired student’s t test with the GraphPad Prism10 software. ns = not significant; ** p ≤ 0.01. (E) Whole cell lysates were prepared, proteins separated by SDS PAGE and subjected to immunoblotting using mAb AC- 15 (β-actin), rabbit pAb MPP8, mouse mAb E1A, mouse mAb E1B-55K, mouse mAb E2A, mouse mAb E4orf6, rat mAb E4orf3, and rabbit pAb Capsid. β-actin serves as a loading control. Molecular weights in kDa are indicated in the left, proteins are indicated on the right. (F) Densitometric analysis of detected band was performed using ImageJ (version 1.54m) to quantify protein levels. Relative protein levels were normalized to the respective housekeeping β-actin steady state levels. Bar charts represent mean values and standard deviations are based on two independent experiments. Statistically significant differences were determined using an unpaired student’s t test with the GraphPad Prism10 software. ns = not significant; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Primary antibodies used for immunoblotting are mouse mAb AC-15 (anti-β-actin; Sigma-Aldrich), mouse mAb MPP8 (sc-398598; Santa Cruz Biotechnology), rabbit pAb MPP8 (HPA040035; Sigma-Aldrich), rabbit pAb PPHLN1 (HPA0038902; Sigma-Aldrich), rabbit pAb TASOR (FAM208A; NBP1-90673; Novus Biologicals), rabbit pAb NP220 (A301-547A; Biomol) mouse mAb Ubiquitin (P4D1; Cell Signaling), rabbit pAb PML (ab72137, abcam), mouse mAb FLAG(M2) (F1804, Sigma Aldrich), mouse mAb E1A (M73; sc-25, Santa Cruz Biotechnology; [ ]), mouse mAb E1B-55K (4E8, [ ]), mouse mAb E2A (B6-8, [ ]), rat mAb E4orf3 (6A11, [ ]), mouse mAb E4orf6 (RSA3, [ ]), and rabbit pAb Capsid (L133, [ ]).

Techniques: Infection, Isolation, Serial Dilution, Virus, Immunofluorescence, Staining, Software, SDS Page, Western Blot, Control, Reverse Transcription

Journal: Cell reports

Article Title: TASOR expression in naive embryonic stem cells safeguards their developmental potential

doi: 10.1016/j.celrep.2024.114887

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: α-TASOR (FAM208A) , Atlas Antibodies , Cat# HPA006735, RRID:AB_1852384.

Techniques: Recombinant, Saline, Staining, Flow Cytometry

Journal: Cell reports

Article Title: TASOR expression in naive embryonic stem cells safeguards their developmental potential

doi: 10.1016/j.celrep.2024.114887

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: α-TASOR (FAM208A) , Atlas Antibodies , Cat# HPA017142, RRID:AB_1852382.

Techniques: Recombinant, Saline, Staining, Flow Cytometry

Journal: Cell reports

Article Title: TASOR expression in naive embryonic stem cells safeguards their developmental potential

doi: 10.1016/j.celrep.2024.114887

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: α-TASOR (FAM208A) , Atlas Antibodies , Cat# HPA006735, RRID:AB_1852384.

Techniques: Recombinant, Saline, Staining, Flow Cytometry