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insect cell line sf21  (Expression Systems Inc)


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    Structured Review

    Expression Systems Inc insect cell line sf21
    A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the <t>Sf21</t> insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In SEC–MALS PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.
    Insect Cell Line Sf21, supplied by Expression Systems Inc, used in various techniques. Bioz Stars score: 99/100, based on 745 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Dimerization of human PARP15 is required for NAD + binding and automodification"

    Article Title: Dimerization of human PARP15 is required for NAD + binding and automodification

    Journal: bioRxiv

    doi: 10.64898/2025.12.15.694324

    A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In SEC–MALS PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.
    Figure Legend Snippet: A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In SEC–MALS PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.

    Techniques Used: Activity Assay, Mutagenesis, Hydrolysis Assay, Modification, Expressing, Incubation, In Vitro, Western Blot, Purification, Control



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    A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the <t>Sf21</t> insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In SEC–MALS PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.
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    A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the <t>Sf21</t> insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In SEC–MALS PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.
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    A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In SEC–MALS PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.

    Journal: bioRxiv

    Article Title: Dimerization of human PARP15 is required for NAD + binding and automodification

    doi: 10.64898/2025.12.15.694324

    Figure Lengend Snippet: A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In SEC–MALS PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.

    Article Snippet: Insect cell line Sf21 was used for the expression of FL protein constructs using Bac-to-Bac® baculovirus expression system (Invitrogen).

    Techniques: Activity Assay, Mutagenesis, Hydrolysis Assay, Modification, Expressing, Incubation, In Vitro, Western Blot, Purification, Control