recombinant protein e2  (Native Antigen Inc)

Journal: bioRxiv

Article Title: Parp7 generates an ADP-ribosyl degron that controls negative feedback of androgen signaling

doi: 10.1101/2024.12.21.629908

Figure Lengend Snippet: DTX2 is the E3 ligase for ADP-ribosylated AR. A, Immunoblot detection of Flag-AR and AR-ADPr (by FL-AF1521) in PC3-AR cells with siRNA knockdowns (total 4-day knockdown) of the selected relevant E3 ligases (DTX1, DTX2, DTX4, HUWE1, RNF146, SPOP, TRIP12 and UBR5), treated with R1881 for 21 hr before cell harvest. The AR/TUB and AR-ADPr/AR ratios for each lane are presented below the blot. B, Immunoblot detection of Flag-AR and AR-ADPr in PC3-AR cells with siDTX2 knockdown, treated with R1881 for times indicated on the panel. The AR-ADPr/AR ratio for each lane is presented below the blot. C, Immunoblot detection of Flag-AR and AR-ADPr in PC3-AR siCTRL and siDTX2 cell extracts and AF1521 bound fraction. Cell extracts from PC3-AR siCTRL and siDTX2 cells treated with R1881 for 6 hr were combined with AF1521 beads for the enrichment of ADP-ribosylated proteins. D, Immunoblot detection of Flag-AR and AR-ADPr in PC3-AR siCTRL/siDTX2 and PC3-AR HA- PARP7 cell extracts and GSH beads bound fraction. Cell extracts from PC3-AR siCTRL/siDTX2 and PC3-AR HA-PARP7 cells treated with R1881 for 6 hr were combined with GSH beads loaded with GST- DTX2-RD or GST-DTX2-RD mut for the enrichment of proteins recognized by DTX2 DTC domain. E, Diagrams of DTX2-RD and DTX2-RD mut . Three loss of function mutations in the DTC domain of DTX2-RD mut (S568A, H582A, and H594A) are indicated with red asterisk. F, Schematic diagram of AR protein preparation as a substrate for biochemical reactions. Cell extracts from PC3-AR siDTX2 cells treated (left) or untreated (right) with R1881 for 6 hr were combined with M2 beads for immunoprecipitation. The purified protein from the preparation with R1881 treatment was used for experiments in panels G and H, and the purified protein from the preparation without R1881 treatment was used only in panel H. G, Immunoblot detection of Flag-AR and AR-ADPr from the ubiquitylation assay on AR protein prepared with siDTX2 and R1881 treatment (panel F, left). The ubiquitylated products (Ub product, red bracket) are labeled for Flag-AR and AR-ADPr detection. All reactions contained AR-ADPr (R1881 treated samples), ATP, Ub, E1 and E2. For DTX2-RD status (dropout or DTX2-RD mut ), refer to labels. H, Immunoblot detection of Flag-AR, AR-ADPr, and GST-DTX2-RD from the ubiquitylation assay on AR protein prepared with siDTX2 transfection, and with or without R1881 treatment (panel F). The ubiquitylated products (Ub product) are labeled in red for Flag-AR and AR-ADPr detection. The dropouts of the ubiquitylation assay components (Ub, E1, E2, and 30°C incubation) are indicated on the labels. The T7-Ubiquitin (T7-Ub) was detected by Ponceau staining. Lane numbers are indicated below the blot. I, Bar plots showing the results of an RT-qPCR experiment in PC3-AR siCTRL/siDTX2 cells untreated (grey), treated with R1881 (purple), and cotreated with R1881 and RBN2397 (blue). The y-axis represents the relative expression normalized to the GUS housekeeping gene, and the x-axis represents the siRNA used. The p-values from the Welch’s t-test for comparisons between corresponding conditions in siCTRL and siDTX2 are indicated on the plots. Error bars represent standard deviation (n = 3; n represents number of biological replicates).

Article Snippet: Ubiquitylation assays were performed at 30°C for 30 min with 1 mM ATP, 100 g/ml Ub (T7-Ub or bovine Ub (Sigma U6253)), 5 g/ml each of UB E1 (R&D Systems E-304) and UB E2 (His-UbcH5C, R&D Systems E2-627), and 20 g/ml GST-DTX2 RD in the buffer E (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM MgCl2, 1 mM DTT, 1 µg/ml each of A/L/P and 0.1 mg/ml BSA).

Techniques: Western Blot, Knockdown, Immunoprecipitation, Purification, Ubiquitin Assay, Labeling, Transfection, Incubation, Staining, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Parp7 generates an ADP-ribosyl degron that controls negative feedback of androgen signaling

doi: 10.1101/2024.12.21.629908

Figure Lengend Snippet: DTX2 conjugates ubiquitin to AR through ADP-ribose A, Immunoblot detection of Fluorescein (FITC) and T7-Ubiquitin (T7-Ub) from the ubiquitylation assay on FITC-AR(C284) or FITC-AR(C284 ADPr ) peptides. The labels indicate from the top: the substrate used (FITC-AR(C284) or FITC-AR(C284 ADPr ) peptides), pre-ubiquitylation assay treatments (NUDT16), the ubiquitylation assay (all reactions contained ATP, T7-Ub, E1 and E2, for DTX2-RD dropout, refer to labels), and the post- ubiquitylation assay treatments (NUDT16 or Mg 2+ buffer). Lane numbers are indicated below the blot. B, Schematic diagram representing FITC-AR(C284 ADPr ) peptide conjugated to ubiquitin (Ub). Indicated with red scissors are bonds within the ADP-ribose structure cleaved by NUDT16 and USP2. C, Schematic of the ubiquitylation assay workflow for panels D and E. D, Immunoblot detection of Flag-AR and AR-ADPr (by FL-AF1521) from the ubiquitylation assay on AR protein prepared with siDTX2 transfection and R1881 treatment (refer to panel 6f for sample preparation workflow). The ubiquitylated products (Ub product) are labeled in red for Flag-AR and AR-ADPr detection. The labels separated by black lines indicate sequential steps from top to bottom. From the top, the labels indicate the substrate used (AR-ADPr), pre-ubiquitylation assay treatments (NUDT16), the ubiquitylation assay, and the post-ubiquitylation assay treatments (USP2-CD, NUDT16, or Mg 2+ buffer). Lane numbers are indicated below the blot. E, Immunoblot detection of Flag-AR and AR-ADPr from the ubiquitylation assay on AR protein prepared with siDTX2 and R1881 treatment (refer to panel 6f for sample preparation workflow). The ubiquitylated products (Ub product) are labeled in red for Flag-AR and AR-ADPr detection. The labels separated by black lines indicate sequential steps from top to bottom. From the top, the labels indicate the substrate used (AR-ADPr), pre-ubiquitylation assay treatments (NUDT16), the ubiquitylation assay, and the post- ubiquitylation assay treatments (USP2-CD, NUDT16, or Mg 2+ buffer). Lane numbers are indicated below the blot. F, Scatter plots depicting the correlation between PARP7 (left) or DTX2 (right) mRNA expression and the response to androgen pathway activity calculated by PARADIGM in primary prostate cancer patients from TCGA-PRAD cohort. Each dot represents one patient (n = 478, n represents number of patients). Pearson correlation coefficients and corresponding p-values are indicated on the plots. G, Kaplan-Meier plot depicting progression-free interval (PFI) in primary prostate cancer patients from the TCGA-PRAD cohort, stratified by PARP7 expression levels. The red line represents patients with high PARP7 expression (top 25%), and the green line represents patients with low PARP7 expression (bottom 25%). The X-axis represents time (days), and the Y-axis represents the progression-free interval probability. The interval distributions were compared using the log-rank test, with the p-value indicating statistical significance. Dotted lines represent the 95% confidence interval.

Article Snippet: Ubiquitylation assays were performed at 30°C for 30 min with 1 mM ATP, 100 g/ml Ub (T7-Ub or bovine Ub (Sigma U6253)), 5 g/ml each of UB E1 (R&D Systems E-304) and UB E2 (His-UbcH5C, R&D Systems E2-627), and 20 g/ml GST-DTX2 RD in the buffer E (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM MgCl2, 1 mM DTT, 1 µg/ml each of A/L/P and 0.1 mg/ml BSA).

Techniques: Western Blot, Ubiquitin Assay, Transfection, Sample Prep, Labeling, Expressing, Activity Assay

Journal: NPJ Vaccines

Article Title: A safe insect-based chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques

doi: 10.1038/s41541-024-01047-z

Figure Lengend Snippet: NHPs were vaccinated with a low (LD) or high doses (HD) of EILV/CHIKV, CHIKV 181/25 or PBS (mock). At day 350, all animals were implanted with electronic data loggers as temperature sensor and challenged subcutaneously with 1.0 × 10 5 PFU of WT La Reunion strain of CHIKV at day 370. A Study design and vaccination timeline [Created with BioRender.com Wang, T. (2023) BioRender.com/o29z240]. B Body temperature changes were recorded every 15 min and reported as mean ± standard error of the mean (SEM) starting 7 days before to 14 days after infection with WT CHIKV strain La Réunion. C Viremia were measured by plaque assays at indicated days post-challenge (PC). UD: Undetectable. Data are presented as means ± SEM.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with CHIKV peptide pools (15 µg/ml), or CHIKV E2 recombinant protein (SinoBiological, USA, 15 ug/ml), EILV/CHIKV (1 × 10 8 PFU/ well), CHIKV VLP (The Native Antigen Company, Oxford, UK, 15 µg/ml), or anti-human Ig capture Ab (Mabtech).

Techniques: Infection

Journal: NPJ Vaccines

Article Title: A safe insect-based chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques

doi: 10.1038/s41541-024-01047-z

Figure Lengend Snippet: A – E Transcriptional changes of PBMCs of vaccinated macaques and mock controls at day 4 PC. A Principal component analysis (PCA) of all normalized transcripts to visualize the relatedness of samples via dimensional reduction. Each dot represents an individual RNA sample for mock (pink; n = 3), EILV/CHIKV (gray; n = 3), and CHIKV 181/25 (aqua; n = 3) groups. Samples that cluster are considered overall more similar, whereas distant samples are considered overall more distinct. B Heatmap of the most differentially expressed mRNAs in each group. Transcripts with a Benjamini–Hochberg adjusted p -value < 0.05 were sorted by log fold change values for EILV/CHIKV-vaccinated compared to CHIKV 181/25-immunized subjects. Red indicates increased expression; blue indicates decreased expression. C Bar plot of the most significant signaling pathways in EILV/CHIKV-vaccinated compared to mock-immunized subjects. Any differentially expressed transcripts with a Benjamini–Hochberg corrected P < 0.1 were deemed significant for enrichment. Pathways were sorted by statistical significance (a higher -log ( P ) indicates higher significance). Red indicates increased expression; blue indicates decreased expression. D Heatmap of the most upregulated pathways in each group; samples were sorted by z-score for EILV/CHIKV-vaccinated compared to CHIKV 181/25-immunized subjects. Yellow/green indicates increased expression, blue/purple indicates decreased expression. E Dot plots depicting predicted cell type trend scores for mock (pink; n = 3), EILV/CHIKV (gray; n = 3), and CHIKV 181/25 (aqua; n = 3) groups. Values were derived from transcriptional signatures indicative for a particular cell subset. F , G PBMCs of day 350 vaccinated NHPs were cultured ex vivo with CHIKV capsid, E3, E2 and E1 peptide pools for 6 h, and stained for IFN-γ, CD3, CD4, or CD8. Total number of IFN-γ + CD4 + and CD8 + T cells is shown. *** P < 0.001, ** P < 0.01, or * P < 0.05 compared to mock. #### P < 0.001, ## P < 0.01, or # P < 0.05 compared to CHIKV 181/25.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with CHIKV peptide pools (15 µg/ml), or CHIKV E2 recombinant protein (SinoBiological, USA, 15 ug/ml), EILV/CHIKV (1 × 10 8 PFU/ well), CHIKV VLP (The Native Antigen Company, Oxford, UK, 15 µg/ml), or anti-human Ig capture Ab (Mabtech).

Techniques: Expressing, Derivative Assay, Cell Culture, Ex Vivo, Staining

Journal: NPJ Vaccines

Article Title: A safe insect-based chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques

doi: 10.1038/s41541-024-01047-z

Figure Lengend Snippet: A – C CHIKV-specific MBC responses by ELISPOT analysis. PBMCs of day 30 ( A ) and day 350 ( B - C ) vaccinated NHPs were stimulated for 5 d with R848 plus rIL-2 and seeded onto ELISPOT plates coated with CHIKV capsid, E3, E2 and E1 peptide pools ( A ), total IgG or CHIKV recombinant E2 protein ( B , C ). A – C Frequencies of CHIKV-specific antibody secreting cells (ASC)s per 10 6 input cells in MBC cultures from the subject. B Images of total IgG-ASCs, CHIKV peptide-specific or CHIKV E2 protein- specific MBCs, are shown. D Serum neutralizing activity against CHIKV 181/25 was measured by a plaque reduction neutralization test (PRNT). E CHIKV E2 binding IgG responses at indicate time points by ELISA. F , G Passive immunization study. Pooled sera collected at day 350 PV of macaques were diluted 1:2 in PBS and transferred to 6 week-old AB6 mice 24 h before infection with a LD100 dose of WT CHIKV. Mice were monitored daily for morbidity. F Survival rate. G Percent weight loss compared to prior infection. *** P < 0.001, ** P < 0.01, or * P < 0.05 compared to mock. #### P < 0.001, ## P < 0.01, or # P < 0.05 compared to CHIKV 181/25.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with CHIKV peptide pools (15 µg/ml), or CHIKV E2 recombinant protein (SinoBiological, USA, 15 ug/ml), EILV/CHIKV (1 × 10 8 PFU/ well), CHIKV VLP (The Native Antigen Company, Oxford, UK, 15 µg/ml), or anti-human Ig capture Ab (Mabtech).

Techniques: Enzyme-linked Immunospot, Recombinant, Activity Assay, Plaque Reduction Neutralization Test, Binding Assay, Enzyme-linked Immunosorbent Assay, Infection

Journal: NPJ Vaccines

Article Title: A safe insect-based chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques

doi: 10.1038/s41541-024-01047-z

Figure Lengend Snippet: NHPs were implanted with electronic data loggers as temperature sensor and bled 14 days before vaccination. NHPs were vaccinated with 1.3 × 10 8 PFU EILV/CHIKV ( n = 3), 5 × 10 5 PFU inactivated WT CHIKV ( n = 3) or PBS (mock, n = 2). At day 6 PV, NHPs were challenged subcutaneously with 10 5 PFU of WT La Reunion strain of CHIKV. A Study design and vaccination timeline [Created with BioRender.com Wang, T. (2023) BioRender.com/o29z240]. B Body temperature changes were recorded every 15 min and reported as hourly mean ± SEM starting 8 days before to 14 days after the challenge with WT CHIKV. C , D Viremia were measured by plaque assays ( C ) or Q-PCR ( D ) at indicated days PC. Undetectable: UD. ** P < 0.01 compared to mock group. Unpaired, 2-tailed Student’s t -test was used to determine the differences. Data are presented as means ± SEM.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with CHIKV peptide pools (15 µg/ml), or CHIKV E2 recombinant protein (SinoBiological, USA, 15 ug/ml), EILV/CHIKV (1 × 10 8 PFU/ well), CHIKV VLP (The Native Antigen Company, Oxford, UK, 15 µg/ml), or anti-human Ig capture Ab (Mabtech).

Techniques:

Journal: NPJ Vaccines

Article Title: A safe insect-based chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques

doi: 10.1038/s41541-024-01047-z

Figure Lengend Snippet: A – D Transcriptional changes of PBMCs in EILV/CHIKV- vaccinated macaques and mock controls at day 7 PV. A PCA of all normalized transcripts to visualize the relatedness of samples via dimensional reduction. Each dot represents an individual RNA sample for mock (pink; n = 3) and EILV/CHIKV (gray; n = 3) groups. Samples that cluster are considered overall more similar, whereas distant samples are considered overall more distinct. B Heatmap of the most differentially expressed mRNAs in EILV/CHIKV-vaccinated compared to the mock-immunized subjects; sorted by statistical significance (Benjamini–Hochberg adjusted P < 0.05). Red indicates increased expression, white indicates no change in expression, blue indicates decreased expression. C Bar plot of the most significant signaling pathways in EILV/CHIKV-vaccinated compared to mock-immunized subjects. Any differentially expressed transcripts with a Benjamini–Hochberg corrected P < 0.1 were deemed significant for enrichment purposes. Pathways are sorted by statistical significance (a higher -log( p -value) indicates higher significance). Red indicates increased expression, blue indicates decreased expression. D Dot plots depicting predicted cell type trend scores for mock (pink; n = 3) and EILV/CHIKV (gray; n = 3) groups. Values were derived from transcriptional signatures indicative for a particular cell subset. E – G Sera cytokines were measured by nonhuman primate inflammation 13-plex kits at indicated time points PC. Data are presented as fold increases compared to the mock group.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with CHIKV peptide pools (15 µg/ml), or CHIKV E2 recombinant protein (SinoBiological, USA, 15 ug/ml), EILV/CHIKV (1 × 10 8 PFU/ well), CHIKV VLP (The Native Antigen Company, Oxford, UK, 15 µg/ml), or anti-human Ig capture Ab (Mabtech).

Techniques: Expressing, Derivative Assay

Journal: NPJ Vaccines

Article Title: A safe insect-based chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques

doi: 10.1038/s41541-024-01047-z

Figure Lengend Snippet: A , B NHPs were vaccinated with EILV/CHIKV, inactivated WT CHIKV or PBS (mock). At day 6 PV, NHPs were challenged subcutaneously with 10 5 PFU of the WT La Reunion strain of CHIKV. At day 4 PC, cells expressing NK cell markers were analyzed. Percent positive ( A ) or total number ( B ) of NK or NK T cells in PBMCs are shown. C Serum neutralizing activity against CHIKV 181/25 was measured by PRNT. D CHIKV E2 binding IgG responses at indicate time points by ELISA. E . ELISPOT quantification of peripheral T cell responses. PBMCs of macaques collected at day 4 PC were stimulated with CHIKV capsid, E3, E2 and E1 peptide pools for 24 h. SFCs were measured by IFN-γ ELISPOT. Data are shown as # of SFC per 10 6 cells. ** P < 0.01, or * P < 0.05 compared to mock. ## P < 0.01 compared to CHIKV 181/25.

Article Snippet: Millipore ELISPOT plates (Millipore Ltd, Darmstadt, Germany) were coated with CHIKV peptide pools (15 µg/ml), or CHIKV E2 recombinant protein (SinoBiological, USA, 15 ug/ml), EILV/CHIKV (1 × 10 8 PFU/ well), CHIKV VLP (The Native Antigen Company, Oxford, UK, 15 µg/ml), or anti-human Ig capture Ab (Mabtech).

Techniques: Expressing, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot