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iv major vault protein mvp  (Danaher Inc)


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    Structured Review

    Danaher Inc iv major vault protein mvp
    Fetal ovarian proteins exhibiting differential expression following continuous or cross-over exposure to chemical cocktails in sewage sludge fertiliser. The accession number is derived from NCBI. Fold change relative to the normalised spot volumes for the CC group are increased if marked “+” and decreased if marked “−“. Fold-change values attaining statistical significance are highlighted in bold. P values are derived by post hoc tests of log-normalised spot volumes. Spots containing significant protein matches that cannot be discriminated between are marked with * next to the spot number. Where the same protein is identified in different spots vertical lines join the spot numbers.
    Iv Major Vault Protein Mvp, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ab145632/TNB%2C+Yellow+chromogenic+standard+for+DTNB+thiol+quantification/pmc03731555-198-50-58
    Average 99 stars, based on 29 article reviews
    iv major vault protein mvp - by Bioz Stars, 2026-09
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    Images

    1) Product Images from "Exposure to chemical cocktails before or after conception – The effect of timing on ovarian development "

    Article Title: Exposure to chemical cocktails before or after conception – The effect of timing on ovarian development

    Journal: Molecular and Cellular Endocrinology

    doi: 10.1016/j.mce.2013.06.016

    Fetal ovarian proteins exhibiting differential expression following continuous or cross-over exposure to chemical cocktails in sewage sludge fertiliser. The accession number is derived from NCBI. Fold change relative to the normalised spot volumes for the CC group are increased if marked “+” and decreased if marked “−“. Fold-change values attaining statistical significance are highlighted in bold. P values are derived by post hoc tests of log-normalised spot volumes. Spots containing significant protein matches that cannot be discriminated between are marked with * next to the spot number. Where the same protein is identified in different spots vertical lines join the spot numbers.
    Figure Legend Snippet: Fetal ovarian proteins exhibiting differential expression following continuous or cross-over exposure to chemical cocktails in sewage sludge fertiliser. The accession number is derived from NCBI. Fold change relative to the normalised spot volumes for the CC group are increased if marked “+” and decreased if marked “−“. Fold-change values attaining statistical significance are highlighted in bold. P values are derived by post hoc tests of log-normalised spot volumes. Spots containing significant protein matches that cannot be discriminated between are marked with * next to the spot number. Where the same protein is identified in different spots vertical lines join the spot numbers.

    Techniques Used: Quantitative Proteomics, Derivative Assay, Activity Assay, Binding Assay, Membrane, Transduction, Conjugation Assay

    Sewage sludge exposure affects MVP, HSP90 and HSP70 proteins in fetal ovaries, quantified by Western blot. (A) Representative bands for each treatment group for each Western blot, including β-actin load control. These bands are all from the same 4 ovaries and all available ovaries were used for these Western blots. (B–F) Quantitation of 5 ovarian proteins shown as box and whisker plots. The band volume for each protein was normalised against β-actin for the same lane (i.e. same ovary) and then expressed relative to the mean normalised band volume of the CC treatment group. The horizontal line in the boxes show the median values, with the limits of the boxes showing the 25% and 75% quantiles and the whiskers showing the 10% and 90% quantiles. Common superscripts between groups, for each protein, denote statistically significant differences at P < 0.05. Where there are no superscripts p values are >0.05. (G) Quantification of MVP immunopositive and immunonegative oocytes shows that there were no significant exposure effects on the density of MVP immunopositive oocytes or the ratio between immunonegative and immunopositive oocytes, although the latter tended to be higher in the CT and TC groups.
    Figure Legend Snippet: Sewage sludge exposure affects MVP, HSP90 and HSP70 proteins in fetal ovaries, quantified by Western blot. (A) Representative bands for each treatment group for each Western blot, including β-actin load control. These bands are all from the same 4 ovaries and all available ovaries were used for these Western blots. (B–F) Quantitation of 5 ovarian proteins shown as box and whisker plots. The band volume for each protein was normalised against β-actin for the same lane (i.e. same ovary) and then expressed relative to the mean normalised band volume of the CC treatment group. The horizontal line in the boxes show the median values, with the limits of the boxes showing the 25% and 75% quantiles and the whiskers showing the 10% and 90% quantiles. Common superscripts between groups, for each protein, denote statistically significant differences at P < 0.05. Where there are no superscripts p values are >0.05. (G) Quantification of MVP immunopositive and immunonegative oocytes shows that there were no significant exposure effects on the density of MVP immunopositive oocytes or the ratio between immunonegative and immunopositive oocytes, although the latter tended to be higher in the CT and TC groups.

    Techniques Used: Western Blot, Control, Quantitation Assay, Whisker Assay

    Networks affected by sewage sludge exposure. Genes shown in bold are those, or their products, identified as significantly altered in at least one treatment group compared with controls (CC) in the present study. Analysis was performed using IPA.
    Figure Legend Snippet: Networks affected by sewage sludge exposure. Genes shown in bold are those, or their products, identified as significantly altered in at least one treatment group compared with controls (CC) in the present study. Analysis was performed using IPA.

    Techniques Used: Ubiquitin Proteomics, Modification

    Related Articles

    Immunofluorescence:

    Article Title: Elamipretide reduces pyroptosis and improves functional recovery after spinal cord injury
    Article Snippet: .. For immunofluorescence staining of cells, the slides with climbed cells were washed and fixed with 4% paraformaldehyde for 20 min, and then permeabilized with 0.1% Triton X‐100 at room temperature for 20 min, followed by closed with sealing fluid for 1 h. Then, the slides were washed and incubated with anti‐NeuN (1:100, Novus, catalog # NBP3‐05554‐100) and anti‐GSDMD (1:100, Abcam, catalog # ab219800) or anti‐NeuN (1:100, Absin, catalog # ab145611) and anti‐NLRP3 (1:100, Absin, catalog # abs151715) and at 4°C overnight. .. Sections were incubated in the secondary antibody at room temperature for 2 h, and then incubated with DAPI (4′,6‐diamidino‐2‐phenylindole) for 10 min. Visualization of slides was performed with a fluorescence microscope (Olympus Inc.).

    Staining:

    Article Title: Elamipretide reduces pyroptosis and improves functional recovery after spinal cord injury
    Article Snippet: .. For immunofluorescence staining of cells, the slides with climbed cells were washed and fixed with 4% paraformaldehyde for 20 min, and then permeabilized with 0.1% Triton X‐100 at room temperature for 20 min, followed by closed with sealing fluid for 1 h. Then, the slides were washed and incubated with anti‐NeuN (1:100, Novus, catalog # NBP3‐05554‐100) and anti‐GSDMD (1:100, Abcam, catalog # ab219800) or anti‐NeuN (1:100, Absin, catalog # ab145611) and anti‐NLRP3 (1:100, Absin, catalog # abs151715) and at 4°C overnight. .. Sections were incubated in the secondary antibody at room temperature for 2 h, and then incubated with DAPI (4′,6‐diamidino‐2‐phenylindole) for 10 min. Visualization of slides was performed with a fluorescence microscope (Olympus Inc.).

    Incubation:

    Article Title: Elamipretide reduces pyroptosis and improves functional recovery after spinal cord injury
    Article Snippet: .. For immunofluorescence staining of cells, the slides with climbed cells were washed and fixed with 4% paraformaldehyde for 20 min, and then permeabilized with 0.1% Triton X‐100 at room temperature for 20 min, followed by closed with sealing fluid for 1 h. Then, the slides were washed and incubated with anti‐NeuN (1:100, Novus, catalog # NBP3‐05554‐100) and anti‐GSDMD (1:100, Abcam, catalog # ab219800) or anti‐NeuN (1:100, Absin, catalog # ab145611) and anti‐NLRP3 (1:100, Absin, catalog # abs151715) and at 4°C overnight. .. Sections were incubated in the secondary antibody at room temperature for 2 h, and then incubated with DAPI (4′,6‐diamidino‐2‐phenylindole) for 10 min. Visualization of slides was performed with a fluorescence microscope (Olympus Inc.).

    Article Title: Elamipretide reduces pyroptosis and improves functional recovery after spinal cord injury.
    Article Snippet: .. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense temperature for 20 min, followed by closed with sealing fluid for 1 h. Then, the slides were washed and incubated with anti- NeuN (1:100, Novus, catalog # NBP3- 05554- 100) and anti- GSDMD (1:100, Abcam, catalog # ab219800) or anti- NeuN (1:100, Absin, catalog # ab145611) and anti- NLRP3 (1:100, Absin, catalog # abs151715) and at 4°C overnight. .. Sections were incubated in the secondary antibody at room temperature for 2 h, and then incubated with DAPI (4′,6- diamidino- 2- phenylindole) for 10 min. Visualization of slides was performed with a fluorescence microscope (Olympus Inc.).

    other:

    Article Title: Identification of Candidate Mitochondrial Inheritance Determinants Using the Mammalian Cell-Free System
    Article Snippet: Rabbit polyclonal anti-PACRG (ab4090), rabbit polyclonal anti-SPATA18 (180154), mouse monoclonal anti-MVP (ab14562) and rabbit polyclonal anti-PSMG2 (ab172909) were purchased from Abcam, Cambridge, United Kingdom.

    Article Title: Identification of candidate mitochondrial inheritance determinants using the mammalian cell-free system
    Article Snippet: Rabbit polyclonal anti-PACRG (ab4090), rabbit polyclonal anti-SPATA18 (180154), mouse monoclonal anti-MVP (ab14562), and rabbit polyclonal anti-PSMG2 (ab172909) were purchased from Abcam, Cambridge, United Kingdom.

    Article Title: Matrix stiffness‐induced α‐tubulin acetylation is required for skin fibrosis formation through activation of Yes‐associated protein
    Article Snippet: The hydrogel was then activated under ultraviolet light for 10 min using 5 mg/ml sulpho‐SANPAH (Cat.#ab145610, Abcam, Cambridge, UK) and washed with 50 mM HEPES twice.

    Article Title: Exploiting the aggregation propensity of beta-lactamases to design inhibitors that induce enzyme misfolding
    Article Snippet: The antibodies and antibiotic product codes used are as follows: monoclonal anti-TEM (Abcam, UK ab12251-8A5A10) 0.5 μg/mL, polyclonal rabbit anti-SHV (custom-made by Eurogentec, Belgium) 1 μg/mL, chicken polyclonal anti-beta Galactosidase (Abcam, ab145634 antibody (ab9361) 2 μg/mL.

    Article Title: Exploiting the aggregation propensity of beta-lactamases to design inhibitors that induce enzyme misfolding
    Article Snippet: The antibiotics used for this study: Penicillin G sodium (Benzylpenicillin sodium, Abcam, catalog # ab145634) 1 μg/mL, Ampicillin (Duchefa Biochemie, Netherlands, A0104.0025), tazobactam sodium salt (Sigma-Aldrich, catalog # T2820-10MG), erythromycin, CAS number 114-07-8 (Sigma-Aldrich, catalog # E5389), chloramphenicol, CAS number 56-75-7 (Duchefa Biochemie), and kanamycin CAS number 56-75-7 (Duchefa Biochemie).

    Centrifugation:

    Article Title: TNF-α-Mediated Endothelial Cell Apoptosis Is Rescued by Hydrogen Sulfide
    Article Snippet: .. The MMTS was removed by pre-cold acetone precipitation at −20 °C for 1 h. After removal of acetone by centrifugation at 13,000× g at 4 °C for 10 min, the proteins were resuspended in HENS buffer (HEN buffer containing 1% SDS) and 4 mM biotin-N-[6-(biotinamido) hexyl]-3′-(2′-pyridyldithio) propinamide (HPDP) (#ab145614, Abcam) at 25 °C for 3 h in the dark. .. Biotinylated proteins were then precipitated by streptavidin-agarose beads (#29200, Thermo Fisher Scientific) (overnight at 4 °C with continuous mixing).



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    (a) SEAKER CAR constructs encoding secreted prodrug-activating enzymes: CPG2–19BBz (CPG2/α-CD19/4–1BB/CD3ζ) and β-Lac-19BBz (β-Lac/α-CD19/4–1BB/CD3ζ). LTR = long terminal repeat, Ψ = psi packaging element, CD8 ss = CD8 signal peptide (gray), HA tag = hemagglutinin epitope tag (yellow), P2A = 2A self cleaving peptide, α-CD19 scFv = CD19-specific single chain variable fragment, 4–1BB = 4–1BB costimulatory domain (blue), CD3ζ = CD3 zeta chain (red). (b) Cytolytic activity of standard 19BBz CAR-T cells, CPG2–19BBz SEAKER cells, and β-Lac-19BBz SEAKER cells against Raji (CD19+) target cells expressing firefly luciferase (18 h, bioluminescence assay; mean ± s.d. of n = 3 biological replicates/sample; representative data from 3 independent donors). (c) Antitumor efficacy of standard 19BBz CAR-T cells, CPG2–19BBz SEAKER cells, and β-Lac-19BBz SEAKER cells, without prodrugs, against Raji xenografts in NSG mice (day 16 post-tumor engraftment, bioluminescent imaging (left) and Kaplan–Meier curve (right). (Log-rank (Mantel–Cox) test: *p<0.05; untreated vs. β-Lac-19BBz: p = 0.003; untreated vs. CPG2–19BBz: p = 0.023; Untreated vs. 19BBz: p = 0.03. Experiment was performed once.) (d) SEAKER enzyme expression in cocultures of anti-CD19 SEAKERS with Raji (CD19+) or SET2 (CD19−) cells (CPG2: ELISA assay; β-Lac: <t>nitrocefin</t> cleavage UV assay; mean ± s.d. of n = 3 biological replicates/samples; Student’s two-tailed t-test: **p<0.01, ***p<0.001; representative of 3 experiments). (e) Trans-cytotoxicity of supernatant fluids from standard 19BBz CAR-T cells and SEAKER cells, with or without the corresponding prodrug, against SET2 target cells (48 h, CellTiter-Glo) (mean ± s.d. of n = 3 biological replicates/samples; Student’s two-tailed t-test: **p<0.01, ***p<0.001).
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    Image Search Results


    (a) SEAKER CAR constructs encoding secreted prodrug-activating enzymes: CPG2–19BBz (CPG2/α-CD19/4–1BB/CD3ζ) and β-Lac-19BBz (β-Lac/α-CD19/4–1BB/CD3ζ). LTR = long terminal repeat, Ψ = psi packaging element, CD8 ss = CD8 signal peptide (gray), HA tag = hemagglutinin epitope tag (yellow), P2A = 2A self cleaving peptide, α-CD19 scFv = CD19-specific single chain variable fragment, 4–1BB = 4–1BB costimulatory domain (blue), CD3ζ = CD3 zeta chain (red). (b) Cytolytic activity of standard 19BBz CAR-T cells, CPG2–19BBz SEAKER cells, and β-Lac-19BBz SEAKER cells against Raji (CD19+) target cells expressing firefly luciferase (18 h, bioluminescence assay; mean ± s.d. of n = 3 biological replicates/sample; representative data from 3 independent donors). (c) Antitumor efficacy of standard 19BBz CAR-T cells, CPG2–19BBz SEAKER cells, and β-Lac-19BBz SEAKER cells, without prodrugs, against Raji xenografts in NSG mice (day 16 post-tumor engraftment, bioluminescent imaging (left) and Kaplan–Meier curve (right). (Log-rank (Mantel–Cox) test: *p<0.05; untreated vs. β-Lac-19BBz: p = 0.003; untreated vs. CPG2–19BBz: p = 0.023; Untreated vs. 19BBz: p = 0.03. Experiment was performed once.) (d) SEAKER enzyme expression in cocultures of anti-CD19 SEAKERS with Raji (CD19+) or SET2 (CD19−) cells (CPG2: ELISA assay; β-Lac: nitrocefin cleavage UV assay; mean ± s.d. of n = 3 biological replicates/samples; Student’s two-tailed t-test: **p<0.01, ***p<0.001; representative of 3 experiments). (e) Trans-cytotoxicity of supernatant fluids from standard 19BBz CAR-T cells and SEAKER cells, with or without the corresponding prodrug, against SET2 target cells (48 h, CellTiter-Glo) (mean ± s.d. of n = 3 biological replicates/samples; Student’s two-tailed t-test: **p<0.01, ***p<0.001).

    Journal: Nature chemical biology

    Article Title: Engineering CAR-T Cells to Activate Small-Molecule Drugs in situ

    doi: 10.1038/s41589-021-00932-1

    Figure Lengend Snippet: (a) SEAKER CAR constructs encoding secreted prodrug-activating enzymes: CPG2–19BBz (CPG2/α-CD19/4–1BB/CD3ζ) and β-Lac-19BBz (β-Lac/α-CD19/4–1BB/CD3ζ). LTR = long terminal repeat, Ψ = psi packaging element, CD8 ss = CD8 signal peptide (gray), HA tag = hemagglutinin epitope tag (yellow), P2A = 2A self cleaving peptide, α-CD19 scFv = CD19-specific single chain variable fragment, 4–1BB = 4–1BB costimulatory domain (blue), CD3ζ = CD3 zeta chain (red). (b) Cytolytic activity of standard 19BBz CAR-T cells, CPG2–19BBz SEAKER cells, and β-Lac-19BBz SEAKER cells against Raji (CD19+) target cells expressing firefly luciferase (18 h, bioluminescence assay; mean ± s.d. of n = 3 biological replicates/sample; representative data from 3 independent donors). (c) Antitumor efficacy of standard 19BBz CAR-T cells, CPG2–19BBz SEAKER cells, and β-Lac-19BBz SEAKER cells, without prodrugs, against Raji xenografts in NSG mice (day 16 post-tumor engraftment, bioluminescent imaging (left) and Kaplan–Meier curve (right). (Log-rank (Mantel–Cox) test: *p<0.05; untreated vs. β-Lac-19BBz: p = 0.003; untreated vs. CPG2–19BBz: p = 0.023; Untreated vs. 19BBz: p = 0.03. Experiment was performed once.) (d) SEAKER enzyme expression in cocultures of anti-CD19 SEAKERS with Raji (CD19+) or SET2 (CD19−) cells (CPG2: ELISA assay; β-Lac: nitrocefin cleavage UV assay; mean ± s.d. of n = 3 biological replicates/samples; Student’s two-tailed t-test: **p<0.01, ***p<0.001; representative of 3 experiments). (e) Trans-cytotoxicity of supernatant fluids from standard 19BBz CAR-T cells and SEAKER cells, with or without the corresponding prodrug, against SET2 target cells (48 h, CellTiter-Glo) (mean ± s.d. of n = 3 biological replicates/samples; Student’s two-tailed t-test: **p<0.01, ***p<0.001).

    Article Snippet: Cell supernatant, mouse ascites, or mouse blood was serially diluted (2-fold) and mixed 1:1 with 0.2 mM nitrocefin (abcam ab145625).

    Techniques: Construct, Activity Assay, Expressing, Luciferase, ATP Bioluminescent Assay, Imaging, Enzyme-linked Immunosorbent Assay, UV Assay, Two Tailed Test

    (a) CPG2 enzyme activity in peripheral blood (left) or peritoneal lavage (right) of Raji tumor-engrafted NSG mice treated with CPG2–19BBz SEAKER cells or β-Lac-19BBz SEAKER cells (negative control), based on methotrexate cleavage assay (fold-change; mean ± s.d. of n = 5 mice per group; Student’s two-tailed t-test: *p<0.05; representative data from 2 independent experiments). (b) β-Lac enzyme activity in peripheral blood (left) or peritoneal lavage (right) of Raji tumor-engrafted mice treated with CPG2–19BBz SEAKER cells (negative control) or β-Lac-19BBz SEAKER cells, based on nitrocefin cleavage assay (fold-change; mean ± s.d. of n = 4 mice per group; Student’s two-tailed t-test: **p<0.01; representative data from 2 independent experiments). (c) Experimental scheme to assess efficacy of SEAKER–prodrug combinations in intraperitoneal tumor model (AMS-Glu (2): 50 mg/kg, ip, bid, 12 doses total; Ceph-AMS (3): 4 mg/kg, ip, bid, 3 doses total). (d,e) Tumor bioluminescence was monitored over time (representative images shown; experiment in panel e was repeated with similar results) (see also Extended Data Fig. 3). (f) Experimental scheme to assess efficacy in a heterogeneous tumor model (2 × 106 total cells, 1:1 Nalm6-mCherry/gLuc (CD19+) and Nalm6-eGFP/fLuc (CD19−). (Ceph-AMS (3): 4 mg/kg, ip, bid, 3 doses total). (g,h) Antitumor efficacy against CD19− Nalm6 cancer cells (fLuc) engrafted within the heterogeneous tumor with CD19+ Nalm6 cancer cells (see also Extended Data Fig. 4), only in mice receiving β-Lac-19BBz SEAKER cells plus Ceph-AMS prodrug (3) (representative images shown on day 17 from 20-day study; one mouse in prodrug-treated group died between day 14 and day 17 and is omitted) (mean ± s.d. of n = 5 mice in SEAKER-treated group and n = 4 mice in SEAKER+prodrug-treated group; Student’s two-tailed t-test: *p<0.05; experiment was performed once). (i) Retention of β-Lac enzyme activity in β-Lac-19BBz SEAKER cells expressing T-cell exhaustion markers (TIM3, LAG3, PD1) after 26 days in Raji-engrafted mice (flow cytometry analysis: FMO = fluorescence minus one control; β-Lac substrate = CCF2-AM) (representative data shown for one of n = 4 mice; representative data from 2 independent experiments).

    Journal: Nature chemical biology

    Article Title: Engineering CAR-T Cells to Activate Small-Molecule Drugs in situ

    doi: 10.1038/s41589-021-00932-1

    Figure Lengend Snippet: (a) CPG2 enzyme activity in peripheral blood (left) or peritoneal lavage (right) of Raji tumor-engrafted NSG mice treated with CPG2–19BBz SEAKER cells or β-Lac-19BBz SEAKER cells (negative control), based on methotrexate cleavage assay (fold-change; mean ± s.d. of n = 5 mice per group; Student’s two-tailed t-test: *p<0.05; representative data from 2 independent experiments). (b) β-Lac enzyme activity in peripheral blood (left) or peritoneal lavage (right) of Raji tumor-engrafted mice treated with CPG2–19BBz SEAKER cells (negative control) or β-Lac-19BBz SEAKER cells, based on nitrocefin cleavage assay (fold-change; mean ± s.d. of n = 4 mice per group; Student’s two-tailed t-test: **p<0.01; representative data from 2 independent experiments). (c) Experimental scheme to assess efficacy of SEAKER–prodrug combinations in intraperitoneal tumor model (AMS-Glu (2): 50 mg/kg, ip, bid, 12 doses total; Ceph-AMS (3): 4 mg/kg, ip, bid, 3 doses total). (d,e) Tumor bioluminescence was monitored over time (representative images shown; experiment in panel e was repeated with similar results) (see also Extended Data Fig. 3). (f) Experimental scheme to assess efficacy in a heterogeneous tumor model (2 × 106 total cells, 1:1 Nalm6-mCherry/gLuc (CD19+) and Nalm6-eGFP/fLuc (CD19−). (Ceph-AMS (3): 4 mg/kg, ip, bid, 3 doses total). (g,h) Antitumor efficacy against CD19− Nalm6 cancer cells (fLuc) engrafted within the heterogeneous tumor with CD19+ Nalm6 cancer cells (see also Extended Data Fig. 4), only in mice receiving β-Lac-19BBz SEAKER cells plus Ceph-AMS prodrug (3) (representative images shown on day 17 from 20-day study; one mouse in prodrug-treated group died between day 14 and day 17 and is omitted) (mean ± s.d. of n = 5 mice in SEAKER-treated group and n = 4 mice in SEAKER+prodrug-treated group; Student’s two-tailed t-test: *p<0.05; experiment was performed once). (i) Retention of β-Lac enzyme activity in β-Lac-19BBz SEAKER cells expressing T-cell exhaustion markers (TIM3, LAG3, PD1) after 26 days in Raji-engrafted mice (flow cytometry analysis: FMO = fluorescence minus one control; β-Lac substrate = CCF2-AM) (representative data shown for one of n = 4 mice; representative data from 2 independent experiments).

    Article Snippet: Cell supernatant, mouse ascites, or mouse blood was serially diluted (2-fold) and mixed 1:1 with 0.2 mM nitrocefin (abcam ab145625).

    Techniques: Activity Assay, Negative Control, Cleavage Assay, Two Tailed Test, Expressing, Flow Cytometry, Fluorescence, Control

    (a) Nitrocefin cleavage-based quantitation of tumor β-Lac concentration in a subcutaneous Raji tumor from a mouse treated with 3 × 106 β-Lac-19BBz SEAKER cells (IV) (representative data from two of n = 5 mice per group in 3 independent experiments). (b) anti-β-Lac immunohistochemistry imaging of subcutaneous Raji tumors extracted on day 15 from mice that were untreated, or received 3 × 106 β-Lac-19BBz SEAKER cells (IV) (top panels – left: isotype control; center: untreated mouse stained with anti-β-Lac antibody; right: β-Lac-19BBz-treated mouse stained with anti-β-Lac antibody). Increased magnification highlights diffuse β-Lac staining throughout the tumor environment (bottom panels) (representative data shown from one of the 3 remaining mice from the n = 5 group used in panel a). (c) Experimental scheme to assess therapeutic efficacy of SEAKER–prodrug combinations in a subcutaneous solid tumor model. Raji tumor cells were engrafted sq on day 0 followed by SEAKER cells iv on day 7. The corresponding prodrug was administered beginning on day 15 (AMS-Glu (2): 50 mg/kg, ip, bid, days 15–20, 12 doses total, or Ceph-AMS (3): 4 mg/kg, ip, bid every other day, days 15, 17, 19, 6 doses total) and mice were monitored for survival. (d) Survival analysis of mice engrafted with subcutaneous Raji tumors receiving subtherapeutic doses of CPG2–19BBz SEAKER cells plus AMS-Glu (2) (left panel), or β-Lac-19BBz SEAKER cells plus Ceph-AMS (3) (right panel). (Arrows denote beginning and end of the prodrug administration period; n = 5 mice/group; log-rank (Mantel–Cox) test: *p<0.05; CPG2–19BBz vs. CPG2–19BBz + AMS-Glu: p = 0.023; β-Lac-19BBz vs. β-Lac-19BBz + Ceph-AMS: p = 0.048; experiment was repeated with similar results).

    Journal: Nature chemical biology

    Article Title: Engineering CAR-T Cells to Activate Small-Molecule Drugs in situ

    doi: 10.1038/s41589-021-00932-1

    Figure Lengend Snippet: (a) Nitrocefin cleavage-based quantitation of tumor β-Lac concentration in a subcutaneous Raji tumor from a mouse treated with 3 × 106 β-Lac-19BBz SEAKER cells (IV) (representative data from two of n = 5 mice per group in 3 independent experiments). (b) anti-β-Lac immunohistochemistry imaging of subcutaneous Raji tumors extracted on day 15 from mice that were untreated, or received 3 × 106 β-Lac-19BBz SEAKER cells (IV) (top panels – left: isotype control; center: untreated mouse stained with anti-β-Lac antibody; right: β-Lac-19BBz-treated mouse stained with anti-β-Lac antibody). Increased magnification highlights diffuse β-Lac staining throughout the tumor environment (bottom panels) (representative data shown from one of the 3 remaining mice from the n = 5 group used in panel a). (c) Experimental scheme to assess therapeutic efficacy of SEAKER–prodrug combinations in a subcutaneous solid tumor model. Raji tumor cells were engrafted sq on day 0 followed by SEAKER cells iv on day 7. The corresponding prodrug was administered beginning on day 15 (AMS-Glu (2): 50 mg/kg, ip, bid, days 15–20, 12 doses total, or Ceph-AMS (3): 4 mg/kg, ip, bid every other day, days 15, 17, 19, 6 doses total) and mice were monitored for survival. (d) Survival analysis of mice engrafted with subcutaneous Raji tumors receiving subtherapeutic doses of CPG2–19BBz SEAKER cells plus AMS-Glu (2) (left panel), or β-Lac-19BBz SEAKER cells plus Ceph-AMS (3) (right panel). (Arrows denote beginning and end of the prodrug administration period; n = 5 mice/group; log-rank (Mantel–Cox) test: *p<0.05; CPG2–19BBz vs. CPG2–19BBz + AMS-Glu: p = 0.023; β-Lac-19BBz vs. β-Lac-19BBz + Ceph-AMS: p = 0.048; experiment was repeated with similar results).

    Article Snippet: Cell supernatant, mouse ascites, or mouse blood was serially diluted (2-fold) and mixed 1:1 with 0.2 mM nitrocefin (abcam ab145625).

    Techniques: Quantitation Assay, Concentration Assay, Immunohistochemistry, Imaging, Control, Staining, Drug discovery

    Assessment of SEAKER cell immunogenicity in an immunocompetent mouse model. (a) Experimental scheme to assess immunogenicity of β-Lac-MUC28z SEAKER cells in a syngeneic intraperitoneal ID8 tumor model. Sera were collected on Days 21, 24, 28, and 31 and tested for anti-β-Lac antibodies in panel b. In a separate experiment, ascites were recovered on Day 28 by peritoneal lavage and tested for the presence of SEAKER cells in panels c,d and β-Lac enzyme activity in panel e. (b) Detection anti-β-Lac antibodies in sera over 10 days following SEAKER cell engraftment (Days 21–31) (median with lines representing each individual mouse of n = 12; experiment was performed once). (c,d) Flow cytometry analysis of SEAKER cell (myc+) persistence among T cells (CD3+) and (e) nitrocefin cleavage-based quantitation of β-Lac enzyme activity in ascites recovered 7 days after SEAKER cell engraftment (Day 28) (representative data shown from n = 2 mice per group; on average, 25% of T cells were SEAKER-positive; experiment was performed once). SSC = side scatter. (f) In a third experiment, mice were treated as in panel a, but without cyclophosphamide pretreatment to maximize the antibody response, then sera were recovered 5 days after SEAKER cell engraftment (Day 26) and analyzed for anti-β-Lac antibodies (n = 1 mouse in untreated group; n = 5 mice in treated group; mean ± s.d. of n = 3 technical replicates from each mouse; experiment was performed once). Sera from the 4 mice showing anti-β-Lac antibodies were used for the ex vivo enzyme activity experiment in panel g. (g) Nitrocefin cleavage-based quantitation of enzyme activity of recombinant β-Lac treated with sera from untreated or SEAKER-treated mice from panel f (n = 2 mice in untreated group; mean ± s.d. of n = 4 mice in treated group; experiment was performed once).

    Journal: Nature chemical biology

    Article Title: Engineering CAR-T Cells to Activate Small-Molecule Drugs in situ

    doi: 10.1038/s41589-021-00932-1

    Figure Lengend Snippet: Assessment of SEAKER cell immunogenicity in an immunocompetent mouse model. (a) Experimental scheme to assess immunogenicity of β-Lac-MUC28z SEAKER cells in a syngeneic intraperitoneal ID8 tumor model. Sera were collected on Days 21, 24, 28, and 31 and tested for anti-β-Lac antibodies in panel b. In a separate experiment, ascites were recovered on Day 28 by peritoneal lavage and tested for the presence of SEAKER cells in panels c,d and β-Lac enzyme activity in panel e. (b) Detection anti-β-Lac antibodies in sera over 10 days following SEAKER cell engraftment (Days 21–31) (median with lines representing each individual mouse of n = 12; experiment was performed once). (c,d) Flow cytometry analysis of SEAKER cell (myc+) persistence among T cells (CD3+) and (e) nitrocefin cleavage-based quantitation of β-Lac enzyme activity in ascites recovered 7 days after SEAKER cell engraftment (Day 28) (representative data shown from n = 2 mice per group; on average, 25% of T cells were SEAKER-positive; experiment was performed once). SSC = side scatter. (f) In a third experiment, mice were treated as in panel a, but without cyclophosphamide pretreatment to maximize the antibody response, then sera were recovered 5 days after SEAKER cell engraftment (Day 26) and analyzed for anti-β-Lac antibodies (n = 1 mouse in untreated group; n = 5 mice in treated group; mean ± s.d. of n = 3 technical replicates from each mouse; experiment was performed once). Sera from the 4 mice showing anti-β-Lac antibodies were used for the ex vivo enzyme activity experiment in panel g. (g) Nitrocefin cleavage-based quantitation of enzyme activity of recombinant β-Lac treated with sera from untreated or SEAKER-treated mice from panel f (n = 2 mice in untreated group; mean ± s.d. of n = 4 mice in treated group; experiment was performed once).

    Article Snippet: Cell supernatant, mouse ascites, or mouse blood was serially diluted (2-fold) and mixed 1:1 with 0.2 mM nitrocefin (abcam ab145625).

    Techniques: Immunopeptidomics, Activity Assay, Flow Cytometry, Quantitation Assay, Ex Vivo, Recombinant