Journal: iScience

Article Title: Novel adenovirus vaccine vectors lacking thrombosis-associated interactions with platelet factor 4

doi: 10.1016/j.isci.2025.114329

Figure Lengend Snippet: Identification of adenovirus and adeno-associated virus vectors lacking binding to PF4 by ELISA-qPCR and aggregate pull-down screening (A) Principle of the ELISA-qPCR technique. Adenovirus (Ad) vector particles (VPs) are allowed to interact with proteins, e.g., PF4, coated on an ELISA plate. After washing, the genomes of VPs that specifically interact with the proteins are released by heating and alkaline treatment and quantified by qPCR. Figure created with BioRender. (B) PF4 binding of vaccine-equivalent vectors. Ad5 was obtained from the Ad-GLN collection. N ≥ 6, two independent repeats. Data are represented as mean ± standard deviation. Two-sided Mann-Whitney U tests. The significance threshold was set at p < 0.05. Significance symbols: ns = non-significant, ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001. (C and D) Screening of human and gorilla Ad (C) and AAV (D) collections for PF4 binding by ELISA-qPCR. For each experiment repeat, the PF4 binding index is computed as described in (Statistics), with positive values indicating significant binding to PF4 and negative values corresponding to overlap in the number of bound VPs in PF4-coated versus control samples. Averages and minimum/maximum range of the PF4 binding index from two to four (Ad5, Ad11, Ad34, and Ad80) independent repeats are displayed. Data are represented as mean ± standard deviation. (E) ELISA-qPCR of Ad5 hexon genetic and chemical variants for PF4 binding. Studied variants include: D151C and T273C point mutations; a 5 kDa polyethylene-glycol (PEG) polymer covalently linked to a cysteine residue, which prevents binding on part of the hexon surface by steric competition; deletion of the HVR1 loop; and the T425A substitution known to ablate the binding of factor X. The E1-deleted, GFP-expressing Ad5 vector was used as control (Ad5). HVR: hyper-variable region. PEG: polyethylene glycol. Schematics created with BioRender. N = 6, two independent repeats. Data are represented as mean ± standard deviation. (F) ELISA-qPCR of selected Ads for binding to mouse PF4. The binding index is computed as described in (Statistics). Measurements were performed once with technical triplicates. Data are represented as mean. (G) Principle of the aggregate pull-down technique. Aggregates forming upon interaction with PF4 are separated from free VPs by low speed centrifugation and titrated by qPCR. Figure created with BioRender. (H) Aggregate pull-down of selected Ads in the absence or presence of PF4. N = 8, two independent repeats. The aggregation rate was calculated based on the titration of Ad genomes both in the pellet (aggregates) and in the supernatant (free VPs). Data are represented as mean ± standard deviation. Two-sided Mann-Whitney U tests. The significance threshold was set at p < 0.05. Significance symbols: ns = non-significant, ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001.

Article Snippet: Mouse PF4 cDNA (SinoBiological #MG50144-M) was cloned under a CMV promoter with CMV enhancer and in front of a bGH poly-A terminator in a pcDNA3.1-C-(k)DYK plasmid backbone ( ).

Techniques: Virus, Binding Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Standard Deviation, MANN-WHITNEY, Control, Polymer, Residue, Expressing, Centrifugation, Titration

Journal: iScience

Article Title: Novel adenovirus vaccine vectors lacking thrombosis-associated interactions with platelet factor 4

doi: 10.1016/j.isci.2025.114329

Figure Lengend Snippet: Surface plasmon resonance identifies different PF4 binding patterns among adenoviruses (A) Surface plasmon resonance (SPR) measurements of PF4 binding to Ad5, Ad34, and Ad5-ΔHVR1. Measurements were conducted in PBS +0.5% BSA +0.005% P20 over 90 s injection time with varying PF4 concentrations. (B) SPR measurements of PF4 binding to indicated Ads were conducted in pure PBS with 600 s injection (grey-colored phase) using a PF4 concentration of 900 nM = 7 μg/mL, followed by 750 s flush. Representative traces are displayed.

Article Snippet: Mouse PF4 cDNA (SinoBiological #MG50144-M) was cloned under a CMV promoter with CMV enhancer and in front of a bGH poly-A terminator in a pcDNA3.1-C-(k)DYK plasmid backbone ( ).

Techniques: SPR Assay, Binding Assay, Injection, Concentration Assay

Journal: iScience

Article Title: Novel adenovirus vaccine vectors lacking thrombosis-associated interactions with platelet factor 4

doi: 10.1016/j.isci.2025.114329

Figure Lengend Snippet: PF4 likely binds to Ad5 hexon HVR1 loop and partially protects Ad5 against neutralizing antibodies (A) Schematic representation of the HVR1 exchange performed to construct the Ad5H34 and Ad34H5 vectors. Figure created with BioRender. (B) ELISA-qPCR of the Ad5H34 and Ad34H5 variants for PF4 binding. Numbers of bound VPs in PF4 coated wells were normalized on the average number from the non-coated wells of the same experiment repeat. N = 8, three independent repeats. Significance levels on top of bars represent the comparison (Mann-Whitney U test) of bound titers with versus without PF4. Pairwise comparisons were also conducted between bound titer ratios of Ad5 versus Ad5H34, and of Ad5 versus Ad34H5. Data are represented as mean ± standard deviation. The significance threshold was set at p < 0.05. Significance symbols: ns = non-significant, ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001. (C) Fraction of PF4 found at given surface-to-surface distance from adenovirus hexons, as sampled from Brownian dynamics (BD) simulations. (D) Distribution of computed surface electrostatic potential as well as integrated values across the whole hexons or hexon surface. (E) Surface potential of Ad particles measured by electrophoretic light scattering (ELS). Data are represented as mean ± standard deviation. N = 3. (F) BD simulations of popular regions for PF4 occupancy on hexons. Structural alignments of Ad34’s hexon and Ad5’s hexon suggest that Ad5’s HVR1 loops protrude more than those of Ad34, potentially enhancing their likelihood to interact with PF4 in the bulk solvent. Detailed molecular images mapping popular PF4 interacting residues to their molecular positions in either Ad34’s hexon or Ad5’s hexon are given in B. (G) PF4 interference assay with Ad5 human serum neutralizing antibodies. Luciferase-expressing Ad5 vectors from the GLN collection were incubated with or without 10 μg/mL PF4 and 1/50 diluted human seronegative serum (“naive serum”) or pooled human intravenous immunoglobulins (“IVIG”) at varying dilutions. A549 cells were then infected with the suspensions at 500 VP/cell (vpc), and luciferase luminescence was measured at 24 hpi. The ratio of luminescence levels between samples with and without PF4 that received identical serum or immunoglobulin treatment is displayed. UT: untreated, without human serum or antibodies. N = 12, four independent repeats. ANOVA test of displayed results yielded p < 0.0001, and Dunnett post-hoc tests were conducted against the “UT” sample for all other samples. Pairwise comparisons that did not yield significant p -values are not displayed on the figure. Data are represented as mean ± standard deviation. The significance threshold was set at p < 0.05. Significance symbols: ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001.

Article Snippet: Mouse PF4 cDNA (SinoBiological #MG50144-M) was cloned under a CMV promoter with CMV enhancer and in front of a bGH poly-A terminator in a pcDNA3.1-C-(k)DYK plasmid backbone ( ).

Techniques: Construct, Enzyme-linked Immunosorbent Assay, Binding Assay, Comparison, MANN-WHITNEY, Standard Deviation, Solvent, Luciferase, Expressing, Incubation, Infection

Journal: iScience

Article Title: Novel adenovirus vaccine vectors lacking thrombosis-associated interactions with platelet factor 4

doi: 10.1016/j.isci.2025.114329

Figure Lengend Snippet: PF4 influences binding Ad infectivity and cell docking with serum and cell type dependency (A–C) For infectivity assays (A–C), VPs were incubated for 10 min at 37°C in optiMEM in the presence or absence of 10 μg/mL PF4, 10% fetal bovine serum (FBS), or human serum seronegative for Ad5, then allowed to infect cultured cells. (A) Fold change in Ad infectivity in A549 cells following Ad incubation with PF4. After infection using 20 vpc, internalized Ad genomes were titrated by qPCR 3 h post-infection (hpi). For each Ad type and each metric, pairwise comparisons were conducted between the samples with and without PF4 using two-sided Mann-Whitney U tests. N ≥ 6, two to five independent repeats. Data are represented as mean ± standard deviation. The significance threshold was set at p < 0.05. Significance symbols: ns = non-significant, ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001. (B) Primary peripheral blood mononuclear cells were infected with 2000 vpc of Ad5 vector from the Ad-GLN collection. Ad-expressed GFP fluorescence was quantified 48 hpi, and the proportions of GFP-positive cells were normalized to the average of the “no PF4, FBS” condition for each cell type. N ≥ 3, one or two independent repeats. Data are represented as mean ± standard deviation. Two-sided Mann-Whitney U tests. The significance threshold was set at p < 0.05. Significance symbols: ns = non-significant, ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001. (C) Primary human nasal epithelium cells were infected with 20 vpc of Ad5 vector from the Ad-GLN collection. Ad-expressed luciferase luminescence was quantified 24 hpi and normalized to the average of the “no PF4, no serum” condition. N ≥ 7. Data are represented as mean ± standard deviation. Two-sided Mann-Whitney U tests. The significance threshold was set at p < 0.05. Significance symbols: ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001. (D) Principle of the erythrocyte pull-down technique. VPs aggregated or docking on erythrocytes, are separated from free VPs by low speed centrifugation and titrated by qPCR. Figure created with BioRender. (E) Erythrocyte pull-down of a fiber-modified Ad5 with ablated CAR tropism (Ad5-ΔCAR) in the absence or presence of PF4. A two-way ANOVA test indicated that both the erythrocytes ( p = 0.00162) and PF4 ( p = 1.15E-8) factors were significant. Pairwise comparisons were performed by two-sided Mann-Whitney U tests. N = 8. Data are represented as mean ± standard deviation. The significance threshold was set at p < 0.05. Significance symbols: ns = non-significant, ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001.

Article Snippet: Mouse PF4 cDNA (SinoBiological #MG50144-M) was cloned under a CMV promoter with CMV enhancer and in front of a bGH poly-A terminator in a pcDNA3.1-C-(k)DYK plasmid backbone ( ).

Techniques: Binding Assay, Infection, Incubation, Cell Culture, MANN-WHITNEY, Standard Deviation, Plasmid Preparation, Fluorescence, Luciferase, Centrifugation, Modification

Journal: iScience

Article Title: Novel adenovirus vaccine vectors lacking thrombosis-associated interactions with platelet factor 4

doi: 10.1016/j.isci.2025.114329

Figure Lengend Snippet: Immunogenicity of PF4 non-binding COVID-19 vaccine candidates in mice (A) Experimental design. Five 10–12 weeks old C57BL/6JCrl male mice per group were immunized intravenously with 5E8 VP of E1-deleted or E1/E3-deleted vectors expressing the S1 domain of the SARS-CoV-2 spike protein (Hu-1 strain). Blood was drawn at 14 and 28 days after immunization (dai), and mice were sacrificed at 30 dai for splenocyte collection. (B–D) (B) Representative FACS plots show the frequencies of peripheral blood S1-epitope specific CD8 + T cells in the different groups (C, D). Percentages of S1-epitope specific CD8 + T cells in blood (C) and numbers in spleen (D) as determined by MHC tetramer staining. Time-course analysis using a mixed model two-way ANOVA was performed in (C). Data are represented as mean ± standard deviation. Two-sided Mann-Whitney U tests. The significance threshold was set at p < 0.05. Significance symbols: ns = non-significant, ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001. (E) Representative FACS plots of stimulated or unstimulated IFN-γ- and TNF-α-secreting CD8 + and CD4 + T cells. (F) Total counts of S1-specific IFN-γ- and TNFα-secreting CD8 + (left) and CD4 + (right) T cells in spleen upon peptide stimulation. Numbers of IFNγ and TNFα secreting cells in non-stimulated control samples were subtracted. Data are represented as mean ± standard deviation. Two-sided Mann-Whitney U tests. The significance threshold was set at p < 0.05. Significance symbols: ns = non-significant, ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001. (G) SARS-CoV-2 neutralizing antibody titer (NT50) in mouse serum. Data are represented as mean ± standard deviation. (H) Vector particle binding antibodies in the serum of immunized mice were determined by ELISA. Each serum was tested against the vector used for the immunization of the respective group. Symbols represent individual mice. LOD: limit of detection. Data are represented as mean ± standard deviation.

Article Snippet: Mouse PF4 cDNA (SinoBiological #MG50144-M) was cloned under a CMV promoter with CMV enhancer and in front of a bGH poly-A terminator in a pcDNA3.1-C-(k)DYK plasmid backbone ( ).

Techniques: Immunopeptidomics, Binding Assay, Expressing, Staining, Standard Deviation, MANN-WHITNEY, Control, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Ecto-5ʹ-nucleotidase/CD73 reduces COX-2 expression in activated macrophages

doi: 10.1038/s41598-025-34809-3

Figure Lengend Snippet: Expression and purification of CD73 protein. ( A ) E. coli Origami cells were transformed with pET28a-CD73 plasmid and plated on LB agar containing kanamycin. Six colonies were randomly picked and PCR performed with primers for CD73. Size of the product is 536 bp. L represents 100 bp plus ladder. ( B ) Transformation was further confirmed in two of the positive colonies through restriction enzyme digestion with EcoRI and XhoI, either singly (SD) or together (DD). Size of the insert (i) is 1731 bp and of vector is 5369 bp. U, undigested plasmid; M, 1 kb ladder. ( C ) SDS-PAGE analysis of the expression of rCD73 was performed from cells treated with different concentrations of IPTG and/or temperature and cultured in a shaker incubator at 300 rpm displayed nearly same level of expression in each case within the pellet. M, Molecular weight marker; S, lysate supernatant; P, lysate pellet. ( D ) The expressed protein was mostly found in the inclusion body (IB) which was washed several times to get a pure IB. ( E ) The protein in the inclusion body was solubilized in 8 M urea. ( F ) Representative Coomassie-stained SDS-PAGE gel shows the various fractions followed by the protein present in the elute in lane E. ( G ) rCD73 was purified using a Ni-NTA column in the AKTA Pure System which showed a single band. ( H ) The denatured rCD73 was refolded in a refolding buffer, concentrated using Amicon Ultracentrifuge filter with a 30-kDa cut-off, and then dialyzed to remove urea. Representative Coomassie-stained SDS-PAGE gel depicts a strong band around 63 kDa. FT, flow through; DF, dialyzed fraction. ( I ) The identity of this band was further confirmed using Western blot tested with antibody against anti-CD73. Lysate from mouse brain was used as a positive control, PC. RP, recombinant protein. ( J ) Absorbance spectra of the refolded protein is depicted in comparison to the denatured protein present in the inclusion body. A shift in the absorbance at 365 nm wavelength (see arrow) indicates protein folding. ( K ) The specific activity of the refolded recombinant protein was tested through phosphatase assay. *** p < 0.001, n = 3.

Article Snippet: The open reading frame of the mouse CD73 gene ( NM_011851.4 , NT5E gene) was commercially obtained as a pMD18-T Simple vector from SinoBiologicals (pMD-mNT5E, Cat. No. MG50231-M, Krishgen Biosystems, Mumbai, India).

Techniques: Expressing, Purification, Transformation Assay, Plasmid Preparation, SDS Page, Cell Culture, Molecular Weight, Marker, Staining, Western Blot, Positive Control, Recombinant, Comparison, Activity Assay, Phosphatase Assay

Journal: Scientific Reports

Article Title: Ecto-5ʹ-nucleotidase/CD73 reduces COX-2 expression in activated macrophages

doi: 10.1038/s41598-025-34809-3

Figure Lengend Snippet: Exogenous addition of recombinant CD73 reduces the effect of ATP on LPS-mediated COX-2 expression. ( A , B ) RAW264.7 macrophage cells were treated with ATP (100 µM), ATPγS (100 µM) or NECA (100 µM), alone or in the presence of LPS (10 ng/ml). Cells were harvested after 24 h and COX-2 expression was checked. β-actin was used as a housekeeping control. ( C ) RAW264.7 cells were treated with different doses of LPS (0.01–10 µg/ml) for 24 h. Culture media was then collected to estimate amount of ATP released. * p < 0.05 with respect to untreated cells ( n = 4). ( D , E ) J774A.1 macrophage cells were treated with LPS (10 ng/ml), alone or together with ATP (100 µM), AMP (100 µM) or NECA (100 µM). 24 h later cells were harvested and checked for COX-2 expression. Representative Western blot image is depicted ( D ) and the relative fold change, calculated by normalization of COX-2 expression with that of β-actin, from n = 3 experiments, is depicted in ( E ). * p < 0.05 with respect to control. $ p < 0.05 with respect to LPS-treated cells. ( F , G ) J774A.1 cells treated with LPS, AMP and NECA, alone or in combination were harvested after 4 h for the isolation of mRNA followed by cDNA synthesis. Real time PCR was performed to check the expression of various M1 ( F ) and M2 ( G ) markers ( n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001 with respect to untreated cells. ( H ) mRNA was isolated from untreated J774A.1 cells and mouse brain tissue and cDNA synthesis was performed. Real time PCR was performed to check the expression of CD73 and CD39 using specific primers. δC t calculated by normalizing with β-actin ( n = 3). ( I , J ) J774A.1 cells were exogenously treated with rCD73 (300 ng) in the presence of ATP alone or ATP together with LPS. 24 h later cell lysates were collected for checking COX-2 expression. Representative blot shown in (I) while relative fold change calculated from normalized COX-2 expression from at least 7 experiments is given in ( J ). ** p < 0.01, *** p < 0.001 ( n = 7). ( K , L ) Cells were treated with LPS, AMP, or AMP-CP (1 µM), in the presence or absence of exogenously added rCD73 protein. After 24 h, cell lysates were used for checking the expression of COX-2. Representative blot shown in ( K ) with relative fold change from normalized COX-2 ( n = 6) given in ( L ). * p < 0.05, ** p < 0.01, *** p < 0.001 relative to the columns shown in the figure. $ p < 0.05 relative to the same stimulation but without rCD73.

Article Snippet: The open reading frame of the mouse CD73 gene ( NM_011851.4 , NT5E gene) was commercially obtained as a pMD18-T Simple vector from SinoBiologicals (pMD-mNT5E, Cat. No. MG50231-M, Krishgen Biosystems, Mumbai, India).

Techniques: Recombinant, Expressing, Control, Western Blot, Isolation, cDNA Synthesis, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Ecto-5ʹ-nucleotidase/CD73 reduces COX-2 expression in activated macrophages

doi: 10.1038/s41598-025-34809-3

Figure Lengend Snippet: Overexpression of CD73 in J774A.1 cells reduces COX-2 expression. ( A ) Presence of the insert (CD73 gene) in the pcDNA3.1 vector was confirmed through PCR ( A ), and restriction analysis using EcoRI and XhoI digestion ( B ). L, 1 kb plus ladder; U, undigested plasmid; SD, EcoRI-treated plasmid; DD, double digestion. Size of the insert, 1731 bp; size of vector, 5446 bp. ( C ) Following transfection, expression of CD73 checked by real time PCR. δC t calculated by normalizing with β-actin. ** p < 0.01 with respect to non-transfected cells ( n = 3). ( D , E ) CD73 expression in transfected cells was analyzed through Western blot, and relative densitometry shown after normalization with β-actin ( n = 4). ( F ) Activity of CD73 in cell lysates was checked by measuring the amount of phosphate released using AMP as a substrate. *** p < 0.001 ( n = 4). ( G ) CD73 activity was also measured in transfected cells ( n = 4). J774A.1 cells were transfected with CD73 for 28 h followed by replacing the media with a phosphate-free DMEM. AMP (100 µM), with or without AMP-CP (1 µM), was then added to the media which was collected after 1 h for the analysis of free phosphate. ( H , I ) J774A.1 cells were transfected with an empty vector (pcDNA3.1) or with CD73 for 28 h followed by stimulation with LPS (10 ng/ml), AMP (100 µM), or AMP-CP (1 µM), as indicated, for 24 h, for analysis of COX-2 protein using Western blot ( H ). Densitometric analysis ( n = 6) represents relative change in COX-2 expression as fold change with respect to empty vector-transfected control cells ( I ). * p < 0.05, ** p < 0.01, *** p < 0.001 with respected to indicated conditions. $$ p < 0.01 with respect to empty vector-transfected cells also treated with LPS and AMP. (J, K) CD73-transfected cells were stimulated with both LPS (10 ng/ml) and ATP (100 µM) for 24 h followed by immunocytochemistry using anti-COX-2 antibody (green). Cells were counterstained with the nuclear dye, Hoechst 33,342 (blue). Scale bar, 20 μm. Integrated density of COX-2 positive signal was normalized with that of Hoechst 33,342 signal and represented as % in (K). *** p < 0.001 ( n = 2).

Article Snippet: The open reading frame of the mouse CD73 gene ( NM_011851.4 , NT5E gene) was commercially obtained as a pMD18-T Simple vector from SinoBiologicals (pMD-mNT5E, Cat. No. MG50231-M, Krishgen Biosystems, Mumbai, India).

Techniques: Over Expression, Expressing, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Control, Immunocytochemistry

Journal: bioRxiv

Article Title: Identifying membrane-bound transcriptional regulatory proteins from rare but evolutionarily conserved domain combinations

doi: 10.64898/2025.12.20.695554

Figure Lengend Snippet: Based on their conserved domain compositions, PREB, CERS2, and NAT8F3 are new candidate MBTRs ( a ) AlphaFold-predicted structures for three potential MBTRs, here showing the mouse proteins. Transmembrane domains are marked in yellow. The domains that comprise the combination with the transmembrane domain are colored blue. ( b ) Phylogenetic trees showing 10 orthologs of each candidate protein with their Pfam domain annotations (top: PREB, middle: CERS2, bottom: NAT8F3 and NAT8F7). The TLC domain includes internal transmembrane domains. The same color scheme as panel ( a ) was applied.

Article Snippet: Mouse Preb cDNA ORF clones were obtained from Sino Biological (MG5A1071-CY) and amplified using primers designed to produce constructs encoding PREB with or without the transmembrane domain (PREBΔTM) ( Supplementary Table S1 ), with the incorporation of Notl and NheI restriction sites (NEB, R3189 and R3131).

Techniques:

Journal: bioRxiv

Article Title: Identifying membrane-bound transcriptional regulatory proteins from rare but evolutionarily conserved domain combinations

doi: 10.64898/2025.12.20.695554

Figure Lengend Snippet: PREB without its transmembrane domain undergoes nuclear translocation and binds to the butyrophilin locus. ( a ) Predicted structure of PREB generated by AlphaFold, highlighting the WD repeat domains, the transmembrane domain, and the site of an engineered C-terminal truncation. ( b ) Schematic of the two constructs created to assay the mouse full-length PREB protein and PREBΔTM, lacking the C-terminal transmembrane domain. The N-terminal biotinylation site is indicated. ( c ) Western blot results of two biological replicates of ES cells expressing PREB and PREBΔTM. Two bands for PREB were detected by Streptavidin-HRP, suggesting C-terminal processing of a full-length PREB when expressed in mouse ES cells. ( d ) Immunofluorescence confocal microscopy images of PREB and PREBΔTM following Dox induction. PREB localizes predominantly to a perinuclear region, forming a ring-like shape. PREBΔTM displays diffused nuclear localization. Rightmost panels show zoomed images corresponding to the boxed regions. ( e ) bioChIP-seq profiles for PREB, PREBΔTM, BirA-only Strep-pulldown, and IgG ChIP-seq control samples. PREBΔTM samples show substantially stronger DNA-binding activity than full-length PREB. ( f ) Representative locus-specific binding profiles of PREB and PREBΔTM at the Btn1a1 (butyrophilin subfamily 1 member A1) gene. PREBΔTM shows markedly higher peak intensities, consistent with enhanced nuclear localization in the absence of the transmembrane domain.

Article Snippet: Mouse Preb cDNA ORF clones were obtained from Sino Biological (MG5A1071-CY) and amplified using primers designed to produce constructs encoding PREB with or without the transmembrane domain (PREBΔTM) ( Supplementary Table S1 ), with the incorporation of Notl and NheI restriction sites (NEB, R3189 and R3131).

Techniques: Translocation Assay, Generated, Construct, Western Blot, Expressing, Immunofluorescence, Confocal Microscopy, ChIP-sequencing, Control, Binding Assay, Activity Assay