dr-gfp 293t cells Search Results


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  • 99
    ATCC hek 293 cells
    Inhibition of NSEP1 protein expression by siRNAs. Western blot assay for detection of NSEP1 (top part) and β-actin (bottom part) from lysates of <t>HEK</t> 293 cells transfected with siRNA duplexes as indicated in the bottom margin: none, no siRNA; GFP,
    Hek 293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 6284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher hek 293 cells
    The Y837F mutation in the NR1 subunit prevents the Csk-induced down-regulation of NMDAR activity. a An example of whole-cell recordings. NMDA-evoked whole-cell currents were recorded from a <t>HEK-293</t> cell co-transfected with cDNAs encoding NR1-1a, NR2A,
    Hek 293 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12989 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa hek 293 cells
    The Y837F mutation in the NR1 subunit prevents the Csk-induced down-regulation of NMDAR activity. a An example of whole-cell recordings. NMDA-evoked whole-cell currents were recorded from a <t>HEK-293</t> cell co-transfected with cDNAs encoding NR1-1a, NR2A,
    Hek 293 Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 514 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa lenti x 293t cells
    The Y837F mutation in the NR1 subunit prevents the Csk-induced down-regulation of NMDAR activity. a An example of whole-cell recordings. NMDA-evoked whole-cell currents were recorded from a <t>HEK-293</t> cell co-transfected with cDNAs encoding NR1-1a, NR2A,
    Lenti X 293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1782 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 293t cells
    The Y837F mutation in the NR1 subunit prevents the Csk-induced down-regulation of NMDAR activity. a An example of whole-cell recordings. NMDA-evoked whole-cell currents were recorded from a <t>HEK-293</t> cell co-transfected with cDNAs encoding NR1-1a, NR2A,
    293t Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7711 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC hek 293t cells
    pp71 induces degradation of exogenous NICD1 and Jag1 proteins via the proteasome in <t>HEK</t> <t>293T</t> cells. pCDH-NICD1 or pCDH-Jag1 was cotransfected with pCDH-GFP, pCDH-flag-UL26, pCDH-pp65, or pCDH-pp71 into HEK 293T cells. Cells were collected at 48 hpt. Overexpression
    Hek 293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 5228 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Roche hek 293 cells
    Comparison of the CMV and EnSM22α promoter-driven shRNAmir constructs potencies in <t>HEK</t> 293 cells. CMV-EGFP-(sh2mir) 2 (lane 2) but not EnSM22α-EGFP-(sh2mir) 2 (lane 4), significantly knocked down exogenous Ca v 1.2 isoform B expression
    Hek 293 Cells, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 2053 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    SignaGen 293t cells
    Comparison of the CMV and EnSM22α promoter-driven shRNAmir constructs potencies in <t>HEK</t> 293 cells. CMV-EGFP-(sh2mir) 2 (lane 2) but not EnSM22α-EGFP-(sh2mir) 2 (lane 4), significantly knocked down exogenous Ca v 1.2 isoform B expression
    293t Cells, supplied by SignaGen, used in various techniques. Bioz Stars score: 92/100, based on 559 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    System Biosciences Inc nih 293t cells
    Comparison of the CMV and EnSM22α promoter-driven shRNAmir constructs potencies in <t>HEK</t> 293 cells. CMV-EGFP-(sh2mir) 2 (lane 2) but not EnSM22α-EGFP-(sh2mir) 2 (lane 4), significantly knocked down exogenous Ca v 1.2 isoform B expression
    Nih 293t Cells, supplied by System Biosciences Inc, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    St. Jude Children's Research Hospital 293t cells
    Comparison of the CMV and EnSM22α promoter-driven shRNAmir constructs potencies in <t>HEK</t> 293 cells. CMV-EGFP-(sh2mir) 2 (lane 2) but not EnSM22α-EGFP-(sh2mir) 2 (lane 4), significantly knocked down exogenous Ca v 1.2 isoform B expression
    293t Cells, supplied by St. Jude Children's Research Hospital, used in various techniques. Bioz Stars score: 93/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Addgene inc 293t cells
    Comparison of the CMV and EnSM22α promoter-driven shRNAmir constructs potencies in <t>HEK</t> 293 cells. CMV-EGFP-(sh2mir) 2 (lane 2) but not EnSM22α-EGFP-(sh2mir) 2 (lane 4), significantly knocked down exogenous Ca v 1.2 isoform B expression
    293t Cells, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 4254 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Stratagene 293t cells
    PHF5A and U2 snRNP component SF3B1 interact with AAV capsid. (A) HeLa or PHF5A-HA-expressing cell lysates were used to pull-down the HA-tagged PHF5A by anti-HA agarose beads. After 15 washes, the HA-tagged PHF5A was detected by anti-HA antibody. (B) Control or PHF5A-HA-expressing HeLa cells were transduced by AAV2 and AAV9 CMV-Luc vectors (MOI 4 x 10 5 ) and total AAV genome copies in the HA pulldown were determined by quantitative real-time PCR. (C) AAV2 CMV-Luc vector (3 x 10 10 genome copies) was unheated or preheated for 30 min at 65°C. PHF5A-HA-over-expressing HeLa cell lysates were then incubated with vectors for 1 hour at 4°C, followed by pulldown of PHF5A-HA. AAV vector genome copies in the precipitates were determined by quantitative real-time PCR. (D) HeLa cells were infected with the AAV2 CMV-Luc vector (4 x 10 10 genome copies/well) for 5 min, 4 or 12 hours. Confocal microscopy analysis was performed to detect the subcellular localizations of AAV vector particles (green) and PHF5A (red). Nuclei were counterstained by DAPI (blue). (E) HeLa cells were infected with AAV2 CMV-Luc vectors (4 x 10 10 genome copies/well) or equivalent amounts of empty AAV2 vectors for 4 hours, and cells were analyzed for co-localization of AAV2 capsid and endogenous PHF5A signals. Prominent co-localized signals were indicated by white arrows. (F) HeLa cells were treated with the PHF5A siRNA for 24 hours, followed by transduction with the AAV2 vector as in E for 4 hours. AAV2 vector particles were detected by anti-AAV2 capsid A20 antibody, and the patterns of cytoplasmic and nuclear accumulations of AAV2 vector particles were compared between control and PHF5A-ablated cells. Representative Z-stack images of the middle sections (slices 3 and 4) from control and PHF5A knockdown cells are shown. (G) Schematic representation for the iodixanol cushion method to enrich cellular factors interacting with particulated AAV capsids. (H) HeLa cell lysates were incubated with AAV2 CMV-Luc vectors (5 x 10 10 genome copies) for 1 hour at 4°C. After centrifugation over 25% iodixanol, three layers (the upper phase, lower phase, and pellet) were separately harvested for Western blotting. AAV capsid proteins VP1, 2 and 3, phospho-SF3B1, and endogenous PHF5A were detected by A20, anti-SF3B1, and anti-PHF5A antibodies, respectively. (I) Same as H for AAV capsid proteins, except that empty AAV2 VP3 only capsids were used for SF3B1 co-precipitation. (J) Control or AAV VP1-over-expressing <t>293T</t> cell lysates were used to pull-down the AAV VP1 protein by A20 antibody. After 15 washes, the pellets were probed for SF3B1 enrichment by anti-SF3B1 antibody.
    293t Cells, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 1156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Mirus Bio 293t cells
    PHF5A and U2 snRNP component SF3B1 interact with AAV capsid. (A) HeLa or PHF5A-HA-expressing cell lysates were used to pull-down the HA-tagged PHF5A by anti-HA agarose beads. After 15 washes, the HA-tagged PHF5A was detected by anti-HA antibody. (B) Control or PHF5A-HA-expressing HeLa cells were transduced by AAV2 and AAV9 CMV-Luc vectors (MOI 4 x 10 5 ) and total AAV genome copies in the HA pulldown were determined by quantitative real-time PCR. (C) AAV2 CMV-Luc vector (3 x 10 10 genome copies) was unheated or preheated for 30 min at 65°C. PHF5A-HA-over-expressing HeLa cell lysates were then incubated with vectors for 1 hour at 4°C, followed by pulldown of PHF5A-HA. AAV vector genome copies in the precipitates were determined by quantitative real-time PCR. (D) HeLa cells were infected with the AAV2 CMV-Luc vector (4 x 10 10 genome copies/well) for 5 min, 4 or 12 hours. Confocal microscopy analysis was performed to detect the subcellular localizations of AAV vector particles (green) and PHF5A (red). Nuclei were counterstained by DAPI (blue). (E) HeLa cells were infected with AAV2 CMV-Luc vectors (4 x 10 10 genome copies/well) or equivalent amounts of empty AAV2 vectors for 4 hours, and cells were analyzed for co-localization of AAV2 capsid and endogenous PHF5A signals. Prominent co-localized signals were indicated by white arrows. (F) HeLa cells were treated with the PHF5A siRNA for 24 hours, followed by transduction with the AAV2 vector as in E for 4 hours. AAV2 vector particles were detected by anti-AAV2 capsid A20 antibody, and the patterns of cytoplasmic and nuclear accumulations of AAV2 vector particles were compared between control and PHF5A-ablated cells. Representative Z-stack images of the middle sections (slices 3 and 4) from control and PHF5A knockdown cells are shown. (G) Schematic representation for the iodixanol cushion method to enrich cellular factors interacting with particulated AAV capsids. (H) HeLa cell lysates were incubated with AAV2 CMV-Luc vectors (5 x 10 10 genome copies) for 1 hour at 4°C. After centrifugation over 25% iodixanol, three layers (the upper phase, lower phase, and pellet) were separately harvested for Western blotting. AAV capsid proteins VP1, 2 and 3, phospho-SF3B1, and endogenous PHF5A were detected by A20, anti-SF3B1, and anti-PHF5A antibodies, respectively. (I) Same as H for AAV capsid proteins, except that empty AAV2 VP3 only capsids were used for SF3B1 co-precipitation. (J) Control or AAV VP1-over-expressing <t>293T</t> cell lysates were used to pull-down the AAV VP1 protein by A20 antibody. After 15 washes, the pellets were probed for SF3B1 enrichment by anti-SF3B1 antibody.
    293t Cells, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 93/100, based on 3248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad 293t cells
    PHF5A and U2 snRNP component SF3B1 interact with AAV capsid. (A) HeLa or PHF5A-HA-expressing cell lysates were used to pull-down the HA-tagged PHF5A by anti-HA agarose beads. After 15 washes, the HA-tagged PHF5A was detected by anti-HA antibody. (B) Control or PHF5A-HA-expressing HeLa cells were transduced by AAV2 and AAV9 CMV-Luc vectors (MOI 4 x 10 5 ) and total AAV genome copies in the HA pulldown were determined by quantitative real-time PCR. (C) AAV2 CMV-Luc vector (3 x 10 10 genome copies) was unheated or preheated for 30 min at 65°C. PHF5A-HA-over-expressing HeLa cell lysates were then incubated with vectors for 1 hour at 4°C, followed by pulldown of PHF5A-HA. AAV vector genome copies in the precipitates were determined by quantitative real-time PCR. (D) HeLa cells were infected with the AAV2 CMV-Luc vector (4 x 10 10 genome copies/well) for 5 min, 4 or 12 hours. Confocal microscopy analysis was performed to detect the subcellular localizations of AAV vector particles (green) and PHF5A (red). Nuclei were counterstained by DAPI (blue). (E) HeLa cells were infected with AAV2 CMV-Luc vectors (4 x 10 10 genome copies/well) or equivalent amounts of empty AAV2 vectors for 4 hours, and cells were analyzed for co-localization of AAV2 capsid and endogenous PHF5A signals. Prominent co-localized signals were indicated by white arrows. (F) HeLa cells were treated with the PHF5A siRNA for 24 hours, followed by transduction with the AAV2 vector as in E for 4 hours. AAV2 vector particles were detected by anti-AAV2 capsid A20 antibody, and the patterns of cytoplasmic and nuclear accumulations of AAV2 vector particles were compared between control and PHF5A-ablated cells. Representative Z-stack images of the middle sections (slices 3 and 4) from control and PHF5A knockdown cells are shown. (G) Schematic representation for the iodixanol cushion method to enrich cellular factors interacting with particulated AAV capsids. (H) HeLa cell lysates were incubated with AAV2 CMV-Luc vectors (5 x 10 10 genome copies) for 1 hour at 4°C. After centrifugation over 25% iodixanol, three layers (the upper phase, lower phase, and pellet) were separately harvested for Western blotting. AAV capsid proteins VP1, 2 and 3, phospho-SF3B1, and endogenous PHF5A were detected by A20, anti-SF3B1, and anti-PHF5A antibodies, respectively. (I) Same as H for AAV capsid proteins, except that empty AAV2 VP3 only capsids were used for SF3B1 co-precipitation. (J) Control or AAV VP1-over-expressing <t>293T</t> cell lysates were used to pull-down the AAV VP1 protein by A20 antibody. After 15 washes, the pellets were probed for SF3B1 enrichment by anti-SF3B1 antibody.
    293t Cells, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Corning Life Sciences 293t cells
    PHF5A and U2 snRNP component SF3B1 interact with AAV capsid. (A) HeLa or PHF5A-HA-expressing cell lysates were used to pull-down the HA-tagged PHF5A by anti-HA agarose beads. After 15 washes, the HA-tagged PHF5A was detected by anti-HA antibody. (B) Control or PHF5A-HA-expressing HeLa cells were transduced by AAV2 and AAV9 CMV-Luc vectors (MOI 4 x 10 5 ) and total AAV genome copies in the HA pulldown were determined by quantitative real-time PCR. (C) AAV2 CMV-Luc vector (3 x 10 10 genome copies) was unheated or preheated for 30 min at 65°C. PHF5A-HA-over-expressing HeLa cell lysates were then incubated with vectors for 1 hour at 4°C, followed by pulldown of PHF5A-HA. AAV vector genome copies in the precipitates were determined by quantitative real-time PCR. (D) HeLa cells were infected with the AAV2 CMV-Luc vector (4 x 10 10 genome copies/well) for 5 min, 4 or 12 hours. Confocal microscopy analysis was performed to detect the subcellular localizations of AAV vector particles (green) and PHF5A (red). Nuclei were counterstained by DAPI (blue). (E) HeLa cells were infected with AAV2 CMV-Luc vectors (4 x 10 10 genome copies/well) or equivalent amounts of empty AAV2 vectors for 4 hours, and cells were analyzed for co-localization of AAV2 capsid and endogenous PHF5A signals. Prominent co-localized signals were indicated by white arrows. (F) HeLa cells were treated with the PHF5A siRNA for 24 hours, followed by transduction with the AAV2 vector as in E for 4 hours. AAV2 vector particles were detected by anti-AAV2 capsid A20 antibody, and the patterns of cytoplasmic and nuclear accumulations of AAV2 vector particles were compared between control and PHF5A-ablated cells. Representative Z-stack images of the middle sections (slices 3 and 4) from control and PHF5A knockdown cells are shown. (G) Schematic representation for the iodixanol cushion method to enrich cellular factors interacting with particulated AAV capsids. (H) HeLa cell lysates were incubated with AAV2 CMV-Luc vectors (5 x 10 10 genome copies) for 1 hour at 4°C. After centrifugation over 25% iodixanol, three layers (the upper phase, lower phase, and pellet) were separately harvested for Western blotting. AAV capsid proteins VP1, 2 and 3, phospho-SF3B1, and endogenous PHF5A were detected by A20, anti-SF3B1, and anti-PHF5A antibodies, respectively. (I) Same as H for AAV capsid proteins, except that empty AAV2 VP3 only capsids were used for SF3B1 co-precipitation. (J) Control or AAV VP1-over-expressing <t>293T</t> cell lysates were used to pull-down the AAV VP1 protein by A20 antibody. After 15 washes, the pellets were probed for SF3B1 enrichment by anti-SF3B1 antibody.
    293t Cells, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 93/100, based on 590 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    TaKaRa hek 293t cells
    PHF5A and U2 snRNP component SF3B1 interact with AAV capsid. (A) HeLa or PHF5A-HA-expressing cell lysates were used to pull-down the HA-tagged PHF5A by anti-HA agarose beads. After 15 washes, the HA-tagged PHF5A was detected by anti-HA antibody. (B) Control or PHF5A-HA-expressing HeLa cells were transduced by AAV2 and AAV9 CMV-Luc vectors (MOI 4 x 10 5 ) and total AAV genome copies in the HA pulldown were determined by quantitative real-time PCR. (C) AAV2 CMV-Luc vector (3 x 10 10 genome copies) was unheated or preheated for 30 min at 65°C. PHF5A-HA-over-expressing HeLa cell lysates were then incubated with vectors for 1 hour at 4°C, followed by pulldown of PHF5A-HA. AAV vector genome copies in the precipitates were determined by quantitative real-time PCR. (D) HeLa cells were infected with the AAV2 CMV-Luc vector (4 x 10 10 genome copies/well) for 5 min, 4 or 12 hours. Confocal microscopy analysis was performed to detect the subcellular localizations of AAV vector particles (green) and PHF5A (red). Nuclei were counterstained by DAPI (blue). (E) HeLa cells were infected with AAV2 CMV-Luc vectors (4 x 10 10 genome copies/well) or equivalent amounts of empty AAV2 vectors for 4 hours, and cells were analyzed for co-localization of AAV2 capsid and endogenous PHF5A signals. Prominent co-localized signals were indicated by white arrows. (F) HeLa cells were treated with the PHF5A siRNA for 24 hours, followed by transduction with the AAV2 vector as in E for 4 hours. AAV2 vector particles were detected by anti-AAV2 capsid A20 antibody, and the patterns of cytoplasmic and nuclear accumulations of AAV2 vector particles were compared between control and PHF5A-ablated cells. Representative Z-stack images of the middle sections (slices 3 and 4) from control and PHF5A knockdown cells are shown. (G) Schematic representation for the iodixanol cushion method to enrich cellular factors interacting with particulated AAV capsids. (H) HeLa cell lysates were incubated with AAV2 CMV-Luc vectors (5 x 10 10 genome copies) for 1 hour at 4°C. After centrifugation over 25% iodixanol, three layers (the upper phase, lower phase, and pellet) were separately harvested for Western blotting. AAV capsid proteins VP1, 2 and 3, phospho-SF3B1, and endogenous PHF5A were detected by A20, anti-SF3B1, and anti-PHF5A antibodies, respectively. (I) Same as H for AAV capsid proteins, except that empty AAV2 VP3 only capsids were used for SF3B1 co-precipitation. (J) Control or AAV VP1-over-expressing <t>293T</t> cell lysates were used to pull-down the AAV VP1 protein by A20 antibody. After 15 washes, the pellets were probed for SF3B1 enrichment by anti-SF3B1 antibody.
    Hek 293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 97/100, based on 684 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche hek 293t cells
    PHF5A and U2 snRNP component SF3B1 interact with AAV capsid. (A) HeLa or PHF5A-HA-expressing cell lysates were used to pull-down the HA-tagged PHF5A by anti-HA agarose beads. After 15 washes, the HA-tagged PHF5A was detected by anti-HA antibody. (B) Control or PHF5A-HA-expressing HeLa cells were transduced by AAV2 and AAV9 CMV-Luc vectors (MOI 4 x 10 5 ) and total AAV genome copies in the HA pulldown were determined by quantitative real-time PCR. (C) AAV2 CMV-Luc vector (3 x 10 10 genome copies) was unheated or preheated for 30 min at 65°C. PHF5A-HA-over-expressing HeLa cell lysates were then incubated with vectors for 1 hour at 4°C, followed by pulldown of PHF5A-HA. AAV vector genome copies in the precipitates were determined by quantitative real-time PCR. (D) HeLa cells were infected with the AAV2 CMV-Luc vector (4 x 10 10 genome copies/well) for 5 min, 4 or 12 hours. Confocal microscopy analysis was performed to detect the subcellular localizations of AAV vector particles (green) and PHF5A (red). Nuclei were counterstained by DAPI (blue). (E) HeLa cells were infected with AAV2 CMV-Luc vectors (4 x 10 10 genome copies/well) or equivalent amounts of empty AAV2 vectors for 4 hours, and cells were analyzed for co-localization of AAV2 capsid and endogenous PHF5A signals. Prominent co-localized signals were indicated by white arrows. (F) HeLa cells were treated with the PHF5A siRNA for 24 hours, followed by transduction with the AAV2 vector as in E for 4 hours. AAV2 vector particles were detected by anti-AAV2 capsid A20 antibody, and the patterns of cytoplasmic and nuclear accumulations of AAV2 vector particles were compared between control and PHF5A-ablated cells. Representative Z-stack images of the middle sections (slices 3 and 4) from control and PHF5A knockdown cells are shown. (G) Schematic representation for the iodixanol cushion method to enrich cellular factors interacting with particulated AAV capsids. (H) HeLa cell lysates were incubated with AAV2 CMV-Luc vectors (5 x 10 10 genome copies) for 1 hour at 4°C. After centrifugation over 25% iodixanol, three layers (the upper phase, lower phase, and pellet) were separately harvested for Western blotting. AAV capsid proteins VP1, 2 and 3, phospho-SF3B1, and endogenous PHF5A were detected by A20, anti-SF3B1, and anti-PHF5A antibodies, respectively. (I) Same as H for AAV capsid proteins, except that empty AAV2 VP3 only capsids were used for SF3B1 co-precipitation. (J) Control or AAV VP1-over-expressing <t>293T</t> cell lysates were used to pull-down the AAV VP1 protein by A20 antibody. After 15 washes, the pellets were probed for SF3B1 enrichment by anti-SF3B1 antibody.
    Hek 293t Cells, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 2057 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Inhibition of NSEP1 protein expression by siRNAs. Western blot assay for detection of NSEP1 (top part) and β-actin (bottom part) from lysates of HEK 293 cells transfected with siRNA duplexes as indicated in the bottom margin: none, no siRNA; GFP,

    Journal:

    Article Title: Nuclease Sensitive Element Binding Protein 1 Associates With the Selenocysteine Insertion Sequence and Functions in Mammalian Selenoprotein Translation

    doi: 10.1002/jcp.20619

    Figure Lengend Snippet: Inhibition of NSEP1 protein expression by siRNAs. Western blot assay for detection of NSEP1 (top part) and β-actin (bottom part) from lysates of HEK 293 cells transfected with siRNA duplexes as indicated in the bottom margin: none, no siRNA; GFP,

    Article Snippet: Twenty-four hours before transfection, approximately 12,000 HEK 293 cells (American Type Culture Collection) were split into each well of 96-well tissue culture plates, with each well containing 100 µl DMEM (high glucose; Invitrogen) with 10% fetal bovine serum and 5 × 10−9 g/ml sodium selenite.

    Techniques: Inhibition, Expressing, Western Blot, Transfection

    Effect of NSEP1 siRNA duplexes on SECIS-dependent luciferase activity in HEK 293 cells. Cells were cotransfected with reporter plasmid pBPHsec (light bars) or pH9A (dark bars) plus siRNA duplexes as indicated in the bottom margin: None, no siRNA; GFP,

    Journal:

    Article Title: Nuclease Sensitive Element Binding Protein 1 Associates With the Selenocysteine Insertion Sequence and Functions in Mammalian Selenoprotein Translation

    doi: 10.1002/jcp.20619

    Figure Lengend Snippet: Effect of NSEP1 siRNA duplexes on SECIS-dependent luciferase activity in HEK 293 cells. Cells were cotransfected with reporter plasmid pBPHsec (light bars) or pH9A (dark bars) plus siRNA duplexes as indicated in the bottom margin: None, no siRNA; GFP,

    Article Snippet: Twenty-four hours before transfection, approximately 12,000 HEK 293 cells (American Type Culture Collection) were split into each well of 96-well tissue culture plates, with each well containing 100 µl DMEM (high glucose; Invitrogen) with 10% fetal bovine serum and 5 × 10−9 g/ml sodium selenite.

    Techniques: Luciferase, Activity Assay, Plasmid Preparation

    The Y837F mutation in the NR1 subunit prevents the Csk-induced down-regulation of NMDAR activity. a An example of whole-cell recordings. NMDA-evoked whole-cell currents were recorded from a HEK-293 cell co-transfected with cDNAs encoding NR1-1a, NR2A,

    Journal: Neurochemical research

    Article Title: The NMDA Receptor NR1 Subunit is Critically Involved in the Regulation of NMDA Receptor Activity by C-terminal Src kinase (Csk)

    doi: 10.1007/s11064-010-0330-0

    Figure Lengend Snippet: The Y837F mutation in the NR1 subunit prevents the Csk-induced down-regulation of NMDAR activity. a An example of whole-cell recordings. NMDA-evoked whole-cell currents were recorded from a HEK-293 cell co-transfected with cDNAs encoding NR1-1a, NR2A,

    Article Snippet: For electrophysiological recordings, cDNAs of green fluorescence protein (GFP, 0.15 μg), NR1-1a or NR1-1a Y837F (0.3 μg), NR2A (1.2 μg) or NR2A (aa 1-857, a gift from Drs. T. Yamamoto and T. Tezuka, Tokyo University, 0.9 μg), n-Src (0.15 μg) and PSD-95 (0.15 μg) were co-transfected with LipoD293™ into HEK-293 cells in culture dishes (diameter: 35 mm). cDNAs encoding the C-tail truncated forms of NR1-1a (aa 1-834) and NR2A (aa 1-837) were amplified by PCR, and then constructed into BamHI - EcoRI sites or EcoRI site of vector pcDNA3 (Invitrogen).

    Techniques: Mutagenesis, Activity Assay, Transfection

    pp71 induces degradation of exogenous NICD1 and Jag1 proteins via the proteasome in HEK 293T cells. pCDH-NICD1 or pCDH-Jag1 was cotransfected with pCDH-GFP, pCDH-flag-UL26, pCDH-pp65, or pCDH-pp71 into HEK 293T cells. Cells were collected at 48 hpt. Overexpression

    Journal: Journal of Virology

    Article Title: Human Cytomegalovirus Infection Dysregulates the Localization and Stability of NICD1 and Jag1 in Neural Progenitor Cells

    doi: 10.1128/JVI.00351-15

    Figure Lengend Snippet: pp71 induces degradation of exogenous NICD1 and Jag1 proteins via the proteasome in HEK 293T cells. pCDH-NICD1 or pCDH-Jag1 was cotransfected with pCDH-GFP, pCDH-flag-UL26, pCDH-pp65, or pCDH-pp71 into HEK 293T cells. Cells were collected at 48 hpt. Overexpression

    Article Snippet: To determine if viral tegument proteins are involved in the degradation of NICD1 and Jag1, plasmids (pCDH-NICD1 and pCDH-Jag1) harboring genes for NICD1 or Jag1 were, respectively, cotransfected with pCDH-GFP (vehicle control), pCDH-flag-UL26, pCDH-pp65, and pCDH-pp71 into HEK 293T cells, which do not express Notch1 and Jag1.

    Techniques: Over Expression

    Capacity of the BRCA1 variants to bind BARD1, PALB2, CtIP and BRIP1. The 3xMyc-tagged BRCA1 proteins were transiently expressed in 293T cells and IPed with an anti-Myc antibody. Note that the amount of CtIP co-IPed with BRCT mutants R1699Q and A1708E was the same as the background level in the vector lane. DOI: http://dx.doi.org/10.7554/eLife.21350.005

    Journal: eLife

    Article Title: Functional and mutational landscapes of BRCA1 for homology-directed repair and therapy resistance

    doi: 10.7554/eLife.21350

    Figure Lengend Snippet: Capacity of the BRCA1 variants to bind BARD1, PALB2, CtIP and BRIP1. The 3xMyc-tagged BRCA1 proteins were transiently expressed in 293T cells and IPed with an anti-Myc antibody. Note that the amount of CtIP co-IPed with BRCT mutants R1699Q and A1708E was the same as the background level in the vector lane. DOI: http://dx.doi.org/10.7554/eLife.21350.005

    Article Snippet: U2OS and 293T cell lines were purchased from American Type Culture Collection (ATCC).

    Techniques: Plasmid Preparation

    Effects of BRCA1 sumoylation and E3 ligase activity on HR and drug resistance. ( A ) Effect of I26A and K119R mutations on BRCA1 binding to BARD1 and other interacting partners. The proteins were transiently expressed in 293T cells and IPed with anti-Myc. ( B ) Quantification of the BARD1-binding capacity of the BRCA1 mutants. Data shown are the means ± SDs of the ratios of BARD1 and BRCA1 band intensities from four independent experiments. **p

    Journal: eLife

    Article Title: Functional and mutational landscapes of BRCA1 for homology-directed repair and therapy resistance

    doi: 10.7554/eLife.21350

    Figure Lengend Snippet: Effects of BRCA1 sumoylation and E3 ligase activity on HR and drug resistance. ( A ) Effect of I26A and K119R mutations on BRCA1 binding to BARD1 and other interacting partners. The proteins were transiently expressed in 293T cells and IPed with anti-Myc. ( B ) Quantification of the BARD1-binding capacity of the BRCA1 mutants. Data shown are the means ± SDs of the ratios of BARD1 and BRCA1 band intensities from four independent experiments. **p

    Article Snippet: U2OS and 293T cell lines were purchased from American Type Culture Collection (ATCC).

    Techniques: Activity Assay, Binding Assay

    Functionalities of BRCA1 deletion mutants in HR, SSA and drug resistance. ( A ) Schematic of wt BRCA1 and 10 overlapping deletions generated for this study. ( B ) Capacity of the deletion mutants in binding key interacting partners. The proteins were transiently expressed in 293T cells and IPed with anti-Myc. ( C ) HR and SSA activities of the deletion mutants. See Figure 1—source data 1 and Figure 2—source data 1 for details. ( D ) Levels of cisplatin and olaparib resistance conferred by the deletion mutants. Values presented are means ± SDs from two to four independent experiments for each mutant. See Figure 3—source data 1 for details. DOI: http://dx.doi.org/10.7554/eLife.21350.015

    Journal: eLife

    Article Title: Functional and mutational landscapes of BRCA1 for homology-directed repair and therapy resistance

    doi: 10.7554/eLife.21350

    Figure Lengend Snippet: Functionalities of BRCA1 deletion mutants in HR, SSA and drug resistance. ( A ) Schematic of wt BRCA1 and 10 overlapping deletions generated for this study. ( B ) Capacity of the deletion mutants in binding key interacting partners. The proteins were transiently expressed in 293T cells and IPed with anti-Myc. ( C ) HR and SSA activities of the deletion mutants. See Figure 1—source data 1 and Figure 2—source data 1 for details. ( D ) Levels of cisplatin and olaparib resistance conferred by the deletion mutants. Values presented are means ± SDs from two to four independent experiments for each mutant. See Figure 3—source data 1 for details. DOI: http://dx.doi.org/10.7554/eLife.21350.015

    Article Snippet: U2OS and 293T cell lines were purchased from American Type Culture Collection (ATCC).

    Techniques: Generated, Binding Assay, Mutagenesis

    IRF4 is a direct transcriptional target of KLF2 in MM cells. ( a ) Microarray analysis in RPMI8226 cells transduced with shKDM3A, shKLF2 or shLuc was performed. The genes significantly downregulated by KDM3A or KLF2 knockdown compared with control were used as gene sets for the GSEA. Normalized enrichment score (NES) and P values are shown. ( b , c ) Quantitative real-time PCR of KLF2 and IRF4 after knockdown ( b ) and overexpression ( c ) of KLF2 in RPMI8226 cells. ( d ) Immunoblot analysis of KLF2 and IRF4 after knockdown of KLF2 in RPMI8226 cells. Shown are the relative signal intensity (shLuc=1) normalized by Actin. Error bars represent s.d. of two independent experiments. ( e ) RPMI8226 cells expressing the KLF2 cDNA carrying synonymous mutations in the shKLF2 #2 target sequence or empty vector were transduced with shKLF2 #2 or shLuc. Whole-cell lysates were subjected to immunoblot analysis. ( f ) Quantitative real-time PCR and immunoblot analysis of KLF2 and IRF4 after knockdown of KLF2 in MM.1S and U266 cells. ( g ) Transactivation of the IRF4 promoter by KLF2. The indicated amounts of KLF2 expression plasmids were transfected into 293T cells together with the human IRF4 promoter-luciferase reporter. The value above each bar indicates the induction level compared with empty vector. ( h ) 293T cells were co-transfected with the indicated IRF4 -luciferase reporter and 0.04 μg of KLF2 expression plasmid or empty vector, and then assayed for luciferase activity. The fold activation (normalized luciferase activity co-transfected with KLF2 expression plasmid relative to empty vector) is shown. ( i ) ChIP analysis showing KLF2 occupancy on IRF4 promoter in RPMI8226 cells. GAPDH promoter was used as negative control. ( j ) KLF2 occupancy is abrogated by KLF2 knockdown on IRF4 promoter in RPMI8226 cells. RPMI8226 cells transduced with either shKLF2 or shLuc were used for ChIP. For b , c , f , i , j , error bars represent s.d. of triplicate measurements. For g , h , data represent mean±s.d. of three ( g ) or two ( h ) biological replicates. For b – j , data are representative of at least two independent experiments. * P

    Journal: Nature Communications

    Article Title: The KDM3A–KLF2–IRF4 axis maintains myeloma cell survival

    doi: 10.1038/ncomms10258

    Figure Lengend Snippet: IRF4 is a direct transcriptional target of KLF2 in MM cells. ( a ) Microarray analysis in RPMI8226 cells transduced with shKDM3A, shKLF2 or shLuc was performed. The genes significantly downregulated by KDM3A or KLF2 knockdown compared with control were used as gene sets for the GSEA. Normalized enrichment score (NES) and P values are shown. ( b , c ) Quantitative real-time PCR of KLF2 and IRF4 after knockdown ( b ) and overexpression ( c ) of KLF2 in RPMI8226 cells. ( d ) Immunoblot analysis of KLF2 and IRF4 after knockdown of KLF2 in RPMI8226 cells. Shown are the relative signal intensity (shLuc=1) normalized by Actin. Error bars represent s.d. of two independent experiments. ( e ) RPMI8226 cells expressing the KLF2 cDNA carrying synonymous mutations in the shKLF2 #2 target sequence or empty vector were transduced with shKLF2 #2 or shLuc. Whole-cell lysates were subjected to immunoblot analysis. ( f ) Quantitative real-time PCR and immunoblot analysis of KLF2 and IRF4 after knockdown of KLF2 in MM.1S and U266 cells. ( g ) Transactivation of the IRF4 promoter by KLF2. The indicated amounts of KLF2 expression plasmids were transfected into 293T cells together with the human IRF4 promoter-luciferase reporter. The value above each bar indicates the induction level compared with empty vector. ( h ) 293T cells were co-transfected with the indicated IRF4 -luciferase reporter and 0.04 μg of KLF2 expression plasmid or empty vector, and then assayed for luciferase activity. The fold activation (normalized luciferase activity co-transfected with KLF2 expression plasmid relative to empty vector) is shown. ( i ) ChIP analysis showing KLF2 occupancy on IRF4 promoter in RPMI8226 cells. GAPDH promoter was used as negative control. ( j ) KLF2 occupancy is abrogated by KLF2 knockdown on IRF4 promoter in RPMI8226 cells. RPMI8226 cells transduced with either shKLF2 or shLuc were used for ChIP. For b , c , f , i , j , error bars represent s.d. of triplicate measurements. For g , h , data represent mean±s.d. of three ( g ) or two ( h ) biological replicates. For b – j , data are representative of at least two independent experiments. * P

    Article Snippet: 293T packing cell line was also obtained from ATCC.

    Techniques: Microarray, Transduction, Real-time Polymerase Chain Reaction, Over Expression, Expressing, Sequencing, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Activation Assay, Chromatin Immunoprecipitation, Negative Control

    Comparison of the CMV and EnSM22α promoter-driven shRNAmir constructs potencies in HEK 293 cells. CMV-EGFP-(sh2mir) 2 (lane 2) but not EnSM22α-EGFP-(sh2mir) 2 (lane 4), significantly knocked down exogenous Ca v 1.2 isoform B expression

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Vascular Smooth Muscle-Specific Knockdown of the Noncardiac Form of the L-Type Calcium Channel by MicroRNA-Based Short Hairpin RNA as a Potential Antihypertensive Therapy

    doi: 10.1124/jpet.108.148866

    Figure Lengend Snippet: Comparison of the CMV and EnSM22α promoter-driven shRNAmir constructs potencies in HEK 293 cells. CMV-EGFP-(sh2mir) 2 (lane 2) but not EnSM22α-EGFP-(sh2mir) 2 (lane 4), significantly knocked down exogenous Ca v 1.2 isoform B expression

    Article Snippet: PC-12 cells (a rat neuroendocrine cell line; American Type Culture Collection) were cultured in RPMI 1640 medium (Invitrogen) supplemented with 2 mM l -glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5g/l glucose, 5% FBS, and 10% horse serum (Invitrogen). siRNA expression constructs were electroporated into A7r5, HL-1, and PC-12 cells with the Cell Line Nucleofector Kit V by Amaxa Nucleofector II (Amaxa Biosystems, Gaithersburg, MD) and cotransfected (1:1) with a rat Cav 1.2 isoform B expression vector (kindly provided by Dr. Sandra Guggino, Johns Hopkins University, Baltimore, MD) into HEK 293 cells using FuGENE 6 (Roche Applied Science, Indianapolis, IN).

    Techniques: Construct, Expressing

    PHF5A and U2 snRNP component SF3B1 interact with AAV capsid. (A) HeLa or PHF5A-HA-expressing cell lysates were used to pull-down the HA-tagged PHF5A by anti-HA agarose beads. After 15 washes, the HA-tagged PHF5A was detected by anti-HA antibody. (B) Control or PHF5A-HA-expressing HeLa cells were transduced by AAV2 and AAV9 CMV-Luc vectors (MOI 4 x 10 5 ) and total AAV genome copies in the HA pulldown were determined by quantitative real-time PCR. (C) AAV2 CMV-Luc vector (3 x 10 10 genome copies) was unheated or preheated for 30 min at 65°C. PHF5A-HA-over-expressing HeLa cell lysates were then incubated with vectors for 1 hour at 4°C, followed by pulldown of PHF5A-HA. AAV vector genome copies in the precipitates were determined by quantitative real-time PCR. (D) HeLa cells were infected with the AAV2 CMV-Luc vector (4 x 10 10 genome copies/well) for 5 min, 4 or 12 hours. Confocal microscopy analysis was performed to detect the subcellular localizations of AAV vector particles (green) and PHF5A (red). Nuclei were counterstained by DAPI (blue). (E) HeLa cells were infected with AAV2 CMV-Luc vectors (4 x 10 10 genome copies/well) or equivalent amounts of empty AAV2 vectors for 4 hours, and cells were analyzed for co-localization of AAV2 capsid and endogenous PHF5A signals. Prominent co-localized signals were indicated by white arrows. (F) HeLa cells were treated with the PHF5A siRNA for 24 hours, followed by transduction with the AAV2 vector as in E for 4 hours. AAV2 vector particles were detected by anti-AAV2 capsid A20 antibody, and the patterns of cytoplasmic and nuclear accumulations of AAV2 vector particles were compared between control and PHF5A-ablated cells. Representative Z-stack images of the middle sections (slices 3 and 4) from control and PHF5A knockdown cells are shown. (G) Schematic representation for the iodixanol cushion method to enrich cellular factors interacting with particulated AAV capsids. (H) HeLa cell lysates were incubated with AAV2 CMV-Luc vectors (5 x 10 10 genome copies) for 1 hour at 4°C. After centrifugation over 25% iodixanol, three layers (the upper phase, lower phase, and pellet) were separately harvested for Western blotting. AAV capsid proteins VP1, 2 and 3, phospho-SF3B1, and endogenous PHF5A were detected by A20, anti-SF3B1, and anti-PHF5A antibodies, respectively. (I) Same as H for AAV capsid proteins, except that empty AAV2 VP3 only capsids were used for SF3B1 co-precipitation. (J) Control or AAV VP1-over-expressing 293T cell lysates were used to pull-down the AAV VP1 protein by A20 antibody. After 15 washes, the pellets were probed for SF3B1 enrichment by anti-SF3B1 antibody.

    Journal: PLoS Pathogens

    Article Title: An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses

    doi: 10.1371/journal.ppat.1005082

    Figure Lengend Snippet: PHF5A and U2 snRNP component SF3B1 interact with AAV capsid. (A) HeLa or PHF5A-HA-expressing cell lysates were used to pull-down the HA-tagged PHF5A by anti-HA agarose beads. After 15 washes, the HA-tagged PHF5A was detected by anti-HA antibody. (B) Control or PHF5A-HA-expressing HeLa cells were transduced by AAV2 and AAV9 CMV-Luc vectors (MOI 4 x 10 5 ) and total AAV genome copies in the HA pulldown were determined by quantitative real-time PCR. (C) AAV2 CMV-Luc vector (3 x 10 10 genome copies) was unheated or preheated for 30 min at 65°C. PHF5A-HA-over-expressing HeLa cell lysates were then incubated with vectors for 1 hour at 4°C, followed by pulldown of PHF5A-HA. AAV vector genome copies in the precipitates were determined by quantitative real-time PCR. (D) HeLa cells were infected with the AAV2 CMV-Luc vector (4 x 10 10 genome copies/well) for 5 min, 4 or 12 hours. Confocal microscopy analysis was performed to detect the subcellular localizations of AAV vector particles (green) and PHF5A (red). Nuclei were counterstained by DAPI (blue). (E) HeLa cells were infected with AAV2 CMV-Luc vectors (4 x 10 10 genome copies/well) or equivalent amounts of empty AAV2 vectors for 4 hours, and cells were analyzed for co-localization of AAV2 capsid and endogenous PHF5A signals. Prominent co-localized signals were indicated by white arrows. (F) HeLa cells were treated with the PHF5A siRNA for 24 hours, followed by transduction with the AAV2 vector as in E for 4 hours. AAV2 vector particles were detected by anti-AAV2 capsid A20 antibody, and the patterns of cytoplasmic and nuclear accumulations of AAV2 vector particles were compared between control and PHF5A-ablated cells. Representative Z-stack images of the middle sections (slices 3 and 4) from control and PHF5A knockdown cells are shown. (G) Schematic representation for the iodixanol cushion method to enrich cellular factors interacting with particulated AAV capsids. (H) HeLa cell lysates were incubated with AAV2 CMV-Luc vectors (5 x 10 10 genome copies) for 1 hour at 4°C. After centrifugation over 25% iodixanol, three layers (the upper phase, lower phase, and pellet) were separately harvested for Western blotting. AAV capsid proteins VP1, 2 and 3, phospho-SF3B1, and endogenous PHF5A were detected by A20, anti-SF3B1, and anti-PHF5A antibodies, respectively. (I) Same as H for AAV capsid proteins, except that empty AAV2 VP3 only capsids were used for SF3B1 co-precipitation. (J) Control or AAV VP1-over-expressing 293T cell lysates were used to pull-down the AAV VP1 protein by A20 antibody. After 15 washes, the pellets were probed for SF3B1 enrichment by anti-SF3B1 antibody.

    Article Snippet: Briefly, 293T cells were transfected with three plasmids, including pHelper (Stratagene), one of the RepCap-expression plasmids (pRep2Cap2, pRep2Cap6, pRep2Cap9, or pRep2Cap8, kindly provided by Dr. James Wilson) and a transfer vector plasmid (pAAV-CMV-Luc, pAAV-SFFV-Luc, pAAV-CMV-Emerald GFP, or pScAAV-CMV-GFP [ ]. pScAAV-CMV-GFP plasmid was kindly provided by Dr. R Jude Samulski through the National Gene Vector Biorepository.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Incubation, Infection, Confocal Microscopy, Transduction, Centrifugation, Western Blot