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  • 92
    Stratagene pcmv tag2b
    Determination of interaction between SARS-CoV N protein and IL-6 promoter. (A) Electrophoretic mobility shift assay (EMSA) was performed with nuclear extracts of A549 cells transfected with (lanes 4, 5, 7, and 8) or without (lanes 1–3, and 6) the N gene. NF-κB probes from nucleotides − 76 to − 63 were labeled and added to the reactions (lanes 1, 2–5, 7, and 8). Unlabeled double-stranded oligonucleotide competitors of NF-κB probe were added during pre-incubation prior to the addition of probes (lanes 2 and 5). For supershift experiments, rabbit anti-N polyclonal antibody (lanes 1 and 4) or normal rabbit polyclonal antibody (rabbit IgG) (lane 8) were incubated with nuclear extracts before adding to the reactions. Samples were electrophoresed on 5% nondenaturing polyacrylamide gel and visualized by autoradiography. Arrows indicate the super-shifted protein–DNA complex, shift band, and free probe. Lane 6: black space. (B) Chromatin immunoprecipitation assay (ChIP) was performed with A549 cells transfected with control <t>pCMV-Tag2B</t> (lane 2) or pCMV-Tag2B-N (lanes 1, 3, 4, and 5). Immunoprecipitated complexes were collected, subjected to PCR amplification, and separated by agarose gel electrophoresis. The IL-6 promoter regions (− 225 to + 13) and (− 76 to + 13) were amplified by PCR using specific primers (IL-6-225U and IL-6-651D or IL-6-76U and IL-6-651D), respectively. Lane 1: input DNA as positive control. Lane 4: normal rabbit polyclonal antibody (rabbit IgG) as a negative control.
    Pcmv Tag2b, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 634 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Agilent technologies pcmv tag2b
    Determination of interaction between SARS-CoV N protein and IL-6 promoter. (A) Electrophoretic mobility shift assay (EMSA) was performed with nuclear extracts of A549 cells transfected with (lanes 4, 5, 7, and 8) or without (lanes 1–3, and 6) the N gene. NF-κB probes from nucleotides − 76 to − 63 were labeled and added to the reactions (lanes 1, 2–5, 7, and 8). Unlabeled double-stranded oligonucleotide competitors of NF-κB probe were added during pre-incubation prior to the addition of probes (lanes 2 and 5). For supershift experiments, rabbit anti-N polyclonal antibody (lanes 1 and 4) or normal rabbit polyclonal antibody (rabbit IgG) (lane 8) were incubated with nuclear extracts before adding to the reactions. Samples were electrophoresed on 5% nondenaturing polyacrylamide gel and visualized by autoradiography. Arrows indicate the super-shifted protein–DNA complex, shift band, and free probe. Lane 6: black space. (B) Chromatin immunoprecipitation assay (ChIP) was performed with A549 cells transfected with control <t>pCMV-Tag2B</t> (lane 2) or pCMV-Tag2B-N (lanes 1, 3, 4, and 5). Immunoprecipitated complexes were collected, subjected to PCR amplification, and separated by agarose gel electrophoresis. The IL-6 promoter regions (− 225 to + 13) and (− 76 to + 13) were amplified by PCR using specific primers (IL-6-225U and IL-6-651D or IL-6-76U and IL-6-651D), respectively. Lane 1: input DNA as positive control. Lane 4: normal rabbit polyclonal antibody (rabbit IgG) as a negative control.
    Pcmv Tag2b, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher pcmv tag2b
    Function of different Danio rerio DDX41 ( Dr DDX41) domains in the signaling pathways. (A) Schematic diagram of full-length and mutated Dr DDX41 fragments, which were inserted into the C-terminus of <t>pCMV-Tag2B.</t> (B,C) Dr DDX41 (B) and its mutants [ (C) , constructed as in panel (A) ] interact with Danio rerio STING ( Dr STING). HEK293T cells were transfected with Flag- Dr DDX41/its mutants and Myc- Dr STING for 24 h. Cell lysates were immunoprecipitated with anti-Myc antibody (Myc) and analyzed by western blot using anti-Flag and anti-Myc antibodies. Expression of the transfected plasmids was analyzed with anti-Flag and anti-Myc antibodies in the whole cell lysates. (D–F) Activation of the nuclear factor-κB (NF-κB)-binding (D) , Hs IRF3 (E) , or Hs IFNβ (F) promoters in HEK293T cells transfected with an NF-κB/ Hs IRF3/ Hs IFNβ-Luc reporter (150 ng/mL) and a renilla luciferase reporter (15 ng/mL) plus different vectors of Dr DDX41 (400 ng/mL) together with pCMV-N-Myc- Dr STING (200 ng/mL) and stimulated with poly(dA:dT) (1 µg/mL) for 6 h. Data are the average luciferase activity ± SD (* p
    Pcmv Tag2b, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Addgene inc pcmv tag2b
    Ectopic EDD increases cisplatin/doxorubicin resistance in MDA-MB-436 cells. (A, B) MDA-MB-436 cells were transfected with increasing concentrations of plasmid <t>pCMV-Tag2B</t> or pCMV-Tag2B.EDD or left untransfected (Con). After 2 days, the cells were harvested for semi-quantitative RT-PCR (A, B; upper panel ) or Western (B; lower panel ) analyses to confirm elevated EDD expression. Each is a representative of 4 experiments. (C, F) MDA-MB-436 cells were treated with increasing doses of cisplatin (C) or doxorubicin (F) for 5 days and cells were counted to determine IC 50 values. Mean ± SEM from 2 independent experiments, each in triplicate. (D, E, G, H) MDA-MB-436 cells were transfected with pCMV-Tag2B or pCMV-Tag2B.EDD. After 24 h (Day 1), the cells were treated ± IC 50 doses of (D, E) cisplatin at 0.3125 μM or (G, H) doxorubicin at 0.023 μM. On Day 5, cells were counted using trypan-blue assay. The results were plotted as (D, G) cell numbers ± drug treatment or (E, H) % cell survival of drug-treated pCMV-Tag2B or pCMV-Tag2B.EDD transfectants, each compared to its untreated control. Mean ± SEM (n = 4-5), *P
    Pcmv Tag2b, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Stratagene pcmv tag2b flag
    Ectopic EDD increases cisplatin/doxorubicin resistance in MDA-MB-436 cells. (A, B) MDA-MB-436 cells were transfected with increasing concentrations of plasmid <t>pCMV-Tag2B</t> or pCMV-Tag2B.EDD or left untransfected (Con). After 2 days, the cells were harvested for semi-quantitative RT-PCR (A, B; upper panel ) or Western (B; lower panel ) analyses to confirm elevated EDD expression. Each is a representative of 4 experiments. (C, F) MDA-MB-436 cells were treated with increasing doses of cisplatin (C) or doxorubicin (F) for 5 days and cells were counted to determine IC 50 values. Mean ± SEM from 2 independent experiments, each in triplicate. (D, E, G, H) MDA-MB-436 cells were transfected with pCMV-Tag2B or pCMV-Tag2B.EDD. After 24 h (Day 1), the cells were treated ± IC 50 doses of (D, E) cisplatin at 0.3125 μM or (G, H) doxorubicin at 0.023 μM. On Day 5, cells were counted using trypan-blue assay. The results were plotted as (D, G) cell numbers ± drug treatment or (E, H) % cell survival of drug-treated pCMV-Tag2B or pCMV-Tag2B.EDD transfectants, each compared to its untreated control. Mean ± SEM (n = 4-5), *P
    Pcmv Tag2b Flag, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcmv tag2b g418
    Ectopic EDD increases cisplatin/doxorubicin resistance in MDA-MB-436 cells. (A, B) MDA-MB-436 cells were transfected with increasing concentrations of plasmid <t>pCMV-Tag2B</t> or pCMV-Tag2B.EDD or left untransfected (Con). After 2 days, the cells were harvested for semi-quantitative RT-PCR (A, B; upper panel ) or Western (B; lower panel ) analyses to confirm elevated EDD expression. Each is a representative of 4 experiments. (C, F) MDA-MB-436 cells were treated with increasing doses of cisplatin (C) or doxorubicin (F) for 5 days and cells were counted to determine IC 50 values. Mean ± SEM from 2 independent experiments, each in triplicate. (D, E, G, H) MDA-MB-436 cells were transfected with pCMV-Tag2B or pCMV-Tag2B.EDD. After 24 h (Day 1), the cells were treated ± IC 50 doses of (D, E) cisplatin at 0.3125 μM or (G, H) doxorubicin at 0.023 μM. On Day 5, cells were counted using trypan-blue assay. The results were plotted as (D, G) cell numbers ± drug treatment or (E, H) % cell survival of drug-treated pCMV-Tag2B or pCMV-Tag2B.EDD transfectants, each compared to its untreated control. Mean ± SEM (n = 4-5), *P
    Pcmv Tag2b G418, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega pcmv tag2b flag
    Ectopic EDD increases cisplatin/doxorubicin resistance in MDA-MB-436 cells. (A, B) MDA-MB-436 cells were transfected with increasing concentrations of plasmid <t>pCMV-Tag2B</t> or pCMV-Tag2B.EDD or left untransfected (Con). After 2 days, the cells were harvested for semi-quantitative RT-PCR (A, B; upper panel ) or Western (B; lower panel ) analyses to confirm elevated EDD expression. Each is a representative of 4 experiments. (C, F) MDA-MB-436 cells were treated with increasing doses of cisplatin (C) or doxorubicin (F) for 5 days and cells were counted to determine IC 50 values. Mean ± SEM from 2 independent experiments, each in triplicate. (D, E, G, H) MDA-MB-436 cells were transfected with pCMV-Tag2B or pCMV-Tag2B.EDD. After 24 h (Day 1), the cells were treated ± IC 50 doses of (D, E) cisplatin at 0.3125 μM or (G, H) doxorubicin at 0.023 μM. On Day 5, cells were counted using trypan-blue assay. The results were plotted as (D, G) cell numbers ± drug treatment or (E, H) % cell survival of drug-treated pCMV-Tag2B or pCMV-Tag2B.EDD transfectants, each compared to its untreated control. Mean ± SEM (n = 4-5), *P
    Pcmv Tag2b Flag, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Addgene inc pcmv tag2b vector
    Ectopic EDD increases cisplatin/doxorubicin resistance in MDA-MB-436 cells. (A, B) MDA-MB-436 cells were transfected with increasing concentrations of plasmid <t>pCMV-Tag2B</t> or pCMV-Tag2B.EDD or left untransfected (Con). After 2 days, the cells were harvested for semi-quantitative RT-PCR (A, B; upper panel ) or Western (B; lower panel ) analyses to confirm elevated EDD expression. Each is a representative of 4 experiments. (C, F) MDA-MB-436 cells were treated with increasing doses of cisplatin (C) or doxorubicin (F) for 5 days and cells were counted to determine IC 50 values. Mean ± SEM from 2 independent experiments, each in triplicate. (D, E, G, H) MDA-MB-436 cells were transfected with pCMV-Tag2B or pCMV-Tag2B.EDD. After 24 h (Day 1), the cells were treated ± IC 50 doses of (D, E) cisplatin at 0.3125 μM or (G, H) doxorubicin at 0.023 μM. On Day 5, cells were counted using trypan-blue assay. The results were plotted as (D, G) cell numbers ± drug treatment or (E, H) % cell survival of drug-treated pCMV-Tag2B or pCMV-Tag2B.EDD transfectants, each compared to its untreated control. Mean ± SEM (n = 4-5), *P
    Pcmv Tag2b Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene pcmv tag2b vector
    Overexpression of KLF2 expression inhibits HepG2 cell proliferation and improves apoptosis. (A) The mRNA level of KLF2 in HepG2 and Hep3B cells transfected with <t>pCMV-Tag2B-KLF2</t> or empty vector was detected by qPCR. (B,C) MTT assays and colony formation assays were used to determine the cell viability for pCMV-Tag2B-KLF2-transfected or empty vector-transfected HepG2 and Hep3B cells. Values represent the mean ± s.d. from three independent experiments. (D) Apoptosis was determined by flow cytometry. UL, necrotic cells; UR, terminal apoptotic cells; LR, early apoptotic cells. * P
    Pcmv Tag2b Vector, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 546 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa pcmv tag2b vector
    The MDM2 3-11-12 s minigene undergoes damage-induced exon 11 skipping in an in vitro splicing system while a control p53 7-8-9 minigene remains unresponsive. ( A ) A minimal MDM2 3-11-12 s minigene, constructed to assess the elements essential for the generation of MDM2-ALT1 alternative splicing, was derived from the previously described MDM2 3-11-12 minigene, which is responsive to stress-induced alternative splicing. The schematic represents the 3-11-12 s minigene and the sizes depicted reflect the length of the exonic and intronic regions of the minigene construct and are inclusive of the Flag-tag and the intervening region (cloning sites) of the <t>pCMV-tag2B</t> vector at the 5′ end of the minigene construct. In vitro -transcribed RNA obtained from the minigenes was subjected to a cell-free in vitro splicing assay using nuclear extracts from either normal (N, NOR) or cisplatinum-treated HeLa S3 cells (C, CIS). RNA was isolated, reversed transcribed and subjected to a 25-cycle PCR using γ- 32 P-radioactively-labeled Flag primer and gene-specific reverse primers. The MDM2 minigene predominantly skips internal exon 11 when spliced in nuclear extracts from cisplatinum-treated cells, but not in nuclear extract from normal cells. The bar graphs represent the percentage of 3.12 skipped product obtained from three independent in vitro splicing experiments under each condition and the error bars represent standard error mean (SEM). The difference in the percentage of 3.12 product between normal and damaged splicing conditions is statistically significant ( n = 3). *Indicates non-specific band also seen in –ATP controls (see Supplementary Figure S1). ∧ Indicates probable PCR degradation products. ( B ) Damage-responsive alternative splicing is transcript-specific. A p53 7-8-9 minigene shows no changes in splicing patterns between the normal and damaged nuclear extract ( n = 3). The sizes of the minigene depicted in the schematic are reflective of the Flag-tag and vector-specific regions at the 5′ end of the minigene construct in a manner similar to the 3-11-12 s minigene.
    Pcmv Tag2b Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agilent technologies pcmv tag2b plasmid
    The interaction with H19 favors the decay-promoting function of KSRP. ( A ) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-KSRP (flag-KSRP), or pTAG2B-H19 (H19) plus pCDNA3-Flag-KSRP (flag-KSRP). Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. ( B ) HEK-293 cells were transiently transfected with GST-fused MS2 binding protein (GST-MS2BP) together with pTAG2B-myog 3′UTR and pTAG2B-MS2-12XH19 (in which murine H19 was tagged with MS2 RNA hairpins; MS2-H19) or pTAG2B-MS2-12X (empty vector; MS2). Total cell extracts were prepared 48 h after transfection and precipitated by glutathione–Sepharose beads. RNA was purified and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. ( C ) HEK-293 cells were transiently cotransfected with <t>pCMV-TAG2B-KSRP</t> (expressing Flag-tagged WT KSRP) or pCMV-TAG2B expressing the indicated Flag-tagged KSRP mutants (KH1GDDG, KH2GDDG, KH3GDDG, KH4GDDG) together with pTAG2B-myog 3′UTR and pTAG2B-H19. Total cell extracts were prepared 48 h after transfection and immunoprecipitated by anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect myogenin 3′UTR (m.Myog 3′UTR) or transfected murine H19 (m.H19). ( D ) In vitro RNA degradation assays using S100 extracts from shKSRP C2C12 cells preincubated (for 90 min at 4 °C) with anti-Flag immunoprecipitates from HEK-293 cells transiently transfected with Flag-tagged WT KSRP (KSRP) or Flag-tagged KH1GDDG mutant (KSRP[KH1GDDG]) together with the E3 sequence or murine H19 cloned in expression vectors. Internally 32 P-labeled and capped RNA substrates were incubated with the aforementioned reaction mixtures, and their decay was monitored for the indicated times. RNA was analyzed by denaturing polyacrylamide gel electrophoresis followed by autoradiography. E3 is a stable transcript used to detect background decay. Representative autoradiograms are displayed. ( E ) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5, or pTAG2B-H19 (H19) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5. Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. The values of RT-qPCR experiments shown are averages (±SEM) of three independent experiments performed in triplicate (* P
    Pcmv Tag2b Plasmid, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Promega pcmv tag2b vector
    The interaction with H19 favors the decay-promoting function of KSRP. ( A ) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-KSRP (flag-KSRP), or pTAG2B-H19 (H19) plus pCDNA3-Flag-KSRP (flag-KSRP). Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. ( B ) HEK-293 cells were transiently transfected with GST-fused MS2 binding protein (GST-MS2BP) together with pTAG2B-myog 3′UTR and pTAG2B-MS2-12XH19 (in which murine H19 was tagged with MS2 RNA hairpins; MS2-H19) or pTAG2B-MS2-12X (empty vector; MS2). Total cell extracts were prepared 48 h after transfection and precipitated by glutathione–Sepharose beads. RNA was purified and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. ( C ) HEK-293 cells were transiently cotransfected with <t>pCMV-TAG2B-KSRP</t> (expressing Flag-tagged WT KSRP) or pCMV-TAG2B expressing the indicated Flag-tagged KSRP mutants (KH1GDDG, KH2GDDG, KH3GDDG, KH4GDDG) together with pTAG2B-myog 3′UTR and pTAG2B-H19. Total cell extracts were prepared 48 h after transfection and immunoprecipitated by anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect myogenin 3′UTR (m.Myog 3′UTR) or transfected murine H19 (m.H19). ( D ) In vitro RNA degradation assays using S100 extracts from shKSRP C2C12 cells preincubated (for 90 min at 4 °C) with anti-Flag immunoprecipitates from HEK-293 cells transiently transfected with Flag-tagged WT KSRP (KSRP) or Flag-tagged KH1GDDG mutant (KSRP[KH1GDDG]) together with the E3 sequence or murine H19 cloned in expression vectors. Internally 32 P-labeled and capped RNA substrates were incubated with the aforementioned reaction mixtures, and their decay was monitored for the indicated times. RNA was analyzed by denaturing polyacrylamide gel electrophoresis followed by autoradiography. E3 is a stable transcript used to detect background decay. Representative autoradiograms are displayed. ( E ) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5, or pTAG2B-H19 (H19) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5. Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. The values of RT-qPCR experiments shown are averages (±SEM) of three independent experiments performed in triplicate (* P
    Pcmv Tag2b Vector, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Roche pcmv tag2b
    The interaction with H19 favors the decay-promoting function of KSRP. ( A ) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-KSRP (flag-KSRP), or pTAG2B-H19 (H19) plus pCDNA3-Flag-KSRP (flag-KSRP). Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. ( B ) HEK-293 cells were transiently transfected with GST-fused MS2 binding protein (GST-MS2BP) together with pTAG2B-myog 3′UTR and pTAG2B-MS2-12XH19 (in which murine H19 was tagged with MS2 RNA hairpins; MS2-H19) or pTAG2B-MS2-12X (empty vector; MS2). Total cell extracts were prepared 48 h after transfection and precipitated by glutathione–Sepharose beads. RNA was purified and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. ( C ) HEK-293 cells were transiently cotransfected with <t>pCMV-TAG2B-KSRP</t> (expressing Flag-tagged WT KSRP) or pCMV-TAG2B expressing the indicated Flag-tagged KSRP mutants (KH1GDDG, KH2GDDG, KH3GDDG, KH4GDDG) together with pTAG2B-myog 3′UTR and pTAG2B-H19. Total cell extracts were prepared 48 h after transfection and immunoprecipitated by anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect myogenin 3′UTR (m.Myog 3′UTR) or transfected murine H19 (m.H19). ( D ) In vitro RNA degradation assays using S100 extracts from shKSRP C2C12 cells preincubated (for 90 min at 4 °C) with anti-Flag immunoprecipitates from HEK-293 cells transiently transfected with Flag-tagged WT KSRP (KSRP) or Flag-tagged KH1GDDG mutant (KSRP[KH1GDDG]) together with the E3 sequence or murine H19 cloned in expression vectors. Internally 32 P-labeled and capped RNA substrates were incubated with the aforementioned reaction mixtures, and their decay was monitored for the indicated times. RNA was analyzed by denaturing polyacrylamide gel electrophoresis followed by autoradiography. E3 is a stable transcript used to detect background decay. Representative autoradiograms are displayed. ( E ) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5, or pTAG2B-H19 (H19) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5. Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. The values of RT-qPCR experiments shown are averages (±SEM) of three independent experiments performed in triplicate (* P
    Pcmv Tag2b, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Stratagene eukaryotic pcmv tag2b expression vector
    The interaction with H19 favors the decay-promoting function of KSRP. ( A ) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-KSRP (flag-KSRP), or pTAG2B-H19 (H19) plus pCDNA3-Flag-KSRP (flag-KSRP). Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. ( B ) HEK-293 cells were transiently transfected with GST-fused MS2 binding protein (GST-MS2BP) together with pTAG2B-myog 3′UTR and pTAG2B-MS2-12XH19 (in which murine H19 was tagged with MS2 RNA hairpins; MS2-H19) or pTAG2B-MS2-12X (empty vector; MS2). Total cell extracts were prepared 48 h after transfection and precipitated by glutathione–Sepharose beads. RNA was purified and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. ( C ) HEK-293 cells were transiently cotransfected with <t>pCMV-TAG2B-KSRP</t> (expressing Flag-tagged WT KSRP) or pCMV-TAG2B expressing the indicated Flag-tagged KSRP mutants (KH1GDDG, KH2GDDG, KH3GDDG, KH4GDDG) together with pTAG2B-myog 3′UTR and pTAG2B-H19. Total cell extracts were prepared 48 h after transfection and immunoprecipitated by anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect myogenin 3′UTR (m.Myog 3′UTR) or transfected murine H19 (m.H19). ( D ) In vitro RNA degradation assays using S100 extracts from shKSRP C2C12 cells preincubated (for 90 min at 4 °C) with anti-Flag immunoprecipitates from HEK-293 cells transiently transfected with Flag-tagged WT KSRP (KSRP) or Flag-tagged KH1GDDG mutant (KSRP[KH1GDDG]) together with the E3 sequence or murine H19 cloned in expression vectors. Internally 32 P-labeled and capped RNA substrates were incubated with the aforementioned reaction mixtures, and their decay was monitored for the indicated times. RNA was analyzed by denaturing polyacrylamide gel electrophoresis followed by autoradiography. E3 is a stable transcript used to detect background decay. Representative autoradiograms are displayed. ( E ) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5, or pTAG2B-H19 (H19) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5. Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. The values of RT-qPCR experiments shown are averages (±SEM) of three independent experiments performed in triplicate (* P
    Eukaryotic Pcmv Tag2b Expression Vector, supplied by Stratagene, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/eukaryotic pcmv tag2b expression vector/product/Stratagene
    Average 91 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
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    91
    Stratagene mammalian expression vector pcmv tag2b
    The interaction with H19 favors the decay-promoting function of KSRP. ( A ) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-KSRP (flag-KSRP), or pTAG2B-H19 (H19) plus pCDNA3-Flag-KSRP (flag-KSRP). Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. ( B ) HEK-293 cells were transiently transfected with GST-fused MS2 binding protein (GST-MS2BP) together with pTAG2B-myog 3′UTR and pTAG2B-MS2-12XH19 (in which murine H19 was tagged with MS2 RNA hairpins; MS2-H19) or pTAG2B-MS2-12X (empty vector; MS2). Total cell extracts were prepared 48 h after transfection and precipitated by glutathione–Sepharose beads. RNA was purified and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. ( C ) HEK-293 cells were transiently cotransfected with <t>pCMV-TAG2B-KSRP</t> (expressing Flag-tagged WT KSRP) or pCMV-TAG2B expressing the indicated Flag-tagged KSRP mutants (KH1GDDG, KH2GDDG, KH3GDDG, KH4GDDG) together with pTAG2B-myog 3′UTR and pTAG2B-H19. Total cell extracts were prepared 48 h after transfection and immunoprecipitated by anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect myogenin 3′UTR (m.Myog 3′UTR) or transfected murine H19 (m.H19). ( D ) In vitro RNA degradation assays using S100 extracts from shKSRP C2C12 cells preincubated (for 90 min at 4 °C) with anti-Flag immunoprecipitates from HEK-293 cells transiently transfected with Flag-tagged WT KSRP (KSRP) or Flag-tagged KH1GDDG mutant (KSRP[KH1GDDG]) together with the E3 sequence or murine H19 cloned in expression vectors. Internally 32 P-labeled and capped RNA substrates were incubated with the aforementioned reaction mixtures, and their decay was monitored for the indicated times. RNA was analyzed by denaturing polyacrylamide gel electrophoresis followed by autoradiography. E3 is a stable transcript used to detect background decay. Representative autoradiograms are displayed. ( E ) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5, or pTAG2B-H19 (H19) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5. Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. The values of RT-qPCR experiments shown are averages (±SEM) of three independent experiments performed in triplicate (* P
    Mammalian Expression Vector Pcmv Tag2b, supplied by Stratagene, used in various techniques. Bioz Stars score: 91/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mammalian expression vector pcmv tag2b/product/Stratagene
    Average 91 stars, based on 47 article reviews
    Price from $9.99 to $1999.99
    mammalian expression vector pcmv tag2b - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

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    Determination of interaction between SARS-CoV N protein and IL-6 promoter. (A) Electrophoretic mobility shift assay (EMSA) was performed with nuclear extracts of A549 cells transfected with (lanes 4, 5, 7, and 8) or without (lanes 1–3, and 6) the N gene. NF-κB probes from nucleotides − 76 to − 63 were labeled and added to the reactions (lanes 1, 2–5, 7, and 8). Unlabeled double-stranded oligonucleotide competitors of NF-κB probe were added during pre-incubation prior to the addition of probes (lanes 2 and 5). For supershift experiments, rabbit anti-N polyclonal antibody (lanes 1 and 4) or normal rabbit polyclonal antibody (rabbit IgG) (lane 8) were incubated with nuclear extracts before adding to the reactions. Samples were electrophoresed on 5% nondenaturing polyacrylamide gel and visualized by autoradiography. Arrows indicate the super-shifted protein–DNA complex, shift band, and free probe. Lane 6: black space. (B) Chromatin immunoprecipitation assay (ChIP) was performed with A549 cells transfected with control pCMV-Tag2B (lane 2) or pCMV-Tag2B-N (lanes 1, 3, 4, and 5). Immunoprecipitated complexes were collected, subjected to PCR amplification, and separated by agarose gel electrophoresis. The IL-6 promoter regions (− 225 to + 13) and (− 76 to + 13) were amplified by PCR using specific primers (IL-6-225U and IL-6-651D or IL-6-76U and IL-6-651D), respectively. Lane 1: input DNA as positive control. Lane 4: normal rabbit polyclonal antibody (rabbit IgG) as a negative control.

    Journal: Virology

    Article Title: Nucleocapsid protein of SARS-CoV activates interleukin-6 expression through cellular transcription factor NF-κB

    doi: 10.1016/j.virol.2007.04.009

    Figure Lengend Snippet: Determination of interaction between SARS-CoV N protein and IL-6 promoter. (A) Electrophoretic mobility shift assay (EMSA) was performed with nuclear extracts of A549 cells transfected with (lanes 4, 5, 7, and 8) or without (lanes 1–3, and 6) the N gene. NF-κB probes from nucleotides − 76 to − 63 were labeled and added to the reactions (lanes 1, 2–5, 7, and 8). Unlabeled double-stranded oligonucleotide competitors of NF-κB probe were added during pre-incubation prior to the addition of probes (lanes 2 and 5). For supershift experiments, rabbit anti-N polyclonal antibody (lanes 1 and 4) or normal rabbit polyclonal antibody (rabbit IgG) (lane 8) were incubated with nuclear extracts before adding to the reactions. Samples were electrophoresed on 5% nondenaturing polyacrylamide gel and visualized by autoradiography. Arrows indicate the super-shifted protein–DNA complex, shift band, and free probe. Lane 6: black space. (B) Chromatin immunoprecipitation assay (ChIP) was performed with A549 cells transfected with control pCMV-Tag2B (lane 2) or pCMV-Tag2B-N (lanes 1, 3, 4, and 5). Immunoprecipitated complexes were collected, subjected to PCR amplification, and separated by agarose gel electrophoresis. The IL-6 promoter regions (− 225 to + 13) and (− 76 to + 13) were amplified by PCR using specific primers (IL-6-225U and IL-6-651D or IL-6-76U and IL-6-651D), respectively. Lane 1: input DNA as positive control. Lane 4: normal rabbit polyclonal antibody (rabbit IgG) as a negative control.

    Article Snippet: All mutant N genes were cloned into Eco RI and Bam HI sites of pCMV-tag2B (Stratagene) to generate plasmids pCMV-tag2B-NΔ220-231, pCMV-tag2B-N1-422, pCMV-tag2B-N1-341, pCMV-tag2B-N61-422S, pCMV-tag2B-N86-422, pCMV-tag2B-N96-42, and pCMV-tag2B-N86-341, respectively.

    Techniques: Electrophoretic Mobility Shift Assay, Transfection, Labeling, Incubation, Autoradiography, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Positive Control, Negative Control

    Functional analysis of deletion mutants of SARS-CoV N protein. (A) Diagram of N protein mutants (left panel) and functional analyses of mutant proteins (right panel). A549 cells were co-transfected with the reporter plasmid pIL6-luc-651 along with plasmid pCMV-Tag2B-N or plasmid containing individual mutant N gene. The effects of each N protein on the activation of IL-6 promoter were determined by measuring luciferase activities of transfected cells. Values correspond to an average of at least three independent experiments. (B) The status of N protein and its mutants expressed in the transfected A549 cells were determined by western bolt using anti-N protein antibodies.

    Journal: Virology

    Article Title: Nucleocapsid protein of SARS-CoV activates interleukin-6 expression through cellular transcription factor NF-κB

    doi: 10.1016/j.virol.2007.04.009

    Figure Lengend Snippet: Functional analysis of deletion mutants of SARS-CoV N protein. (A) Diagram of N protein mutants (left panel) and functional analyses of mutant proteins (right panel). A549 cells were co-transfected with the reporter plasmid pIL6-luc-651 along with plasmid pCMV-Tag2B-N or plasmid containing individual mutant N gene. The effects of each N protein on the activation of IL-6 promoter were determined by measuring luciferase activities of transfected cells. Values correspond to an average of at least three independent experiments. (B) The status of N protein and its mutants expressed in the transfected A549 cells were determined by western bolt using anti-N protein antibodies.

    Article Snippet: All mutant N genes were cloned into Eco RI and Bam HI sites of pCMV-tag2B (Stratagene) to generate plasmids pCMV-tag2B-NΔ220-231, pCMV-tag2B-N1-422, pCMV-tag2B-N1-341, pCMV-tag2B-N61-422S, pCMV-tag2B-N86-422, pCMV-tag2B-N96-42, and pCMV-tag2B-N86-341, respectively.

    Techniques: Functional Assay, Mutagenesis, Transfection, Plasmid Preparation, Activation Assay, Luciferase, Western Blot

    Determination of roles of SARS-CoV N protein in the activation of IL-6 gene. (A) A549 cells were co-transfected with plasmid pCMV-tag2B-N, pCMV-tag2B-M, pCMV-tag2B-S, or pCMV-tag2B-E expressing N, M, S, or E protein, respectively, along with the reporter plasmid pIL6-luc-651 in which the luciferase gene is under the control of IL-6 promoter. The negative control is parental plasmid pCMV-tag2B. Relative luciferase activity was determined by standard procedures. (B) The expression of N, S, M, or E protein in transfected A549 cells was evaluated by western bolt analysis using anti-flag antibody. (C) A549 cells were co-transfected with different amount of plasmid pCMV-Tag2B-N as indicated along with the reporter plasmid pIL6-luc-651. Relative luciferase activities were determined from transfected cells by standard procedures. (D) A549 cells were transfected with plasmid pCMV-Tag2B-N or pCMV-Tag2B. Supernatants of transfected cells were prepared and concentration of IL-6 protein was determined by ELISA using anti-IL-6 antibody. (E) A549 cells (lane 1) were transfected with pCMV-Tag2B (lane 2) or pCMV-Tag2B-N (lane 3). The levels of IL-6 mRNA expressed in transfected cells were determined by RT-PCR using primers specific to IL-6 and the levels of β-actin mRNA produced were measured using primers specific to β-actin. All product bands were visualized and quantified using Gel-Pro Analyzer. The results were expressed as the mean + S.D. of three independent experiments performed in triplicate.

    Journal: Virology

    Article Title: Nucleocapsid protein of SARS-CoV activates interleukin-6 expression through cellular transcription factor NF-κB

    doi: 10.1016/j.virol.2007.04.009

    Figure Lengend Snippet: Determination of roles of SARS-CoV N protein in the activation of IL-6 gene. (A) A549 cells were co-transfected with plasmid pCMV-tag2B-N, pCMV-tag2B-M, pCMV-tag2B-S, or pCMV-tag2B-E expressing N, M, S, or E protein, respectively, along with the reporter plasmid pIL6-luc-651 in which the luciferase gene is under the control of IL-6 promoter. The negative control is parental plasmid pCMV-tag2B. Relative luciferase activity was determined by standard procedures. (B) The expression of N, S, M, or E protein in transfected A549 cells was evaluated by western bolt analysis using anti-flag antibody. (C) A549 cells were co-transfected with different amount of plasmid pCMV-Tag2B-N as indicated along with the reporter plasmid pIL6-luc-651. Relative luciferase activities were determined from transfected cells by standard procedures. (D) A549 cells were transfected with plasmid pCMV-Tag2B-N or pCMV-Tag2B. Supernatants of transfected cells were prepared and concentration of IL-6 protein was determined by ELISA using anti-IL-6 antibody. (E) A549 cells (lane 1) were transfected with pCMV-Tag2B (lane 2) or pCMV-Tag2B-N (lane 3). The levels of IL-6 mRNA expressed in transfected cells were determined by RT-PCR using primers specific to IL-6 and the levels of β-actin mRNA produced were measured using primers specific to β-actin. All product bands were visualized and quantified using Gel-Pro Analyzer. The results were expressed as the mean + S.D. of three independent experiments performed in triplicate.

    Article Snippet: All mutant N genes were cloned into Eco RI and Bam HI sites of pCMV-tag2B (Stratagene) to generate plasmids pCMV-tag2B-NΔ220-231, pCMV-tag2B-N1-422, pCMV-tag2B-N1-341, pCMV-tag2B-N61-422S, pCMV-tag2B-N86-422, pCMV-tag2B-N96-42, and pCMV-tag2B-N86-341, respectively.

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Negative Control, Activity Assay, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Produced

    Functional analysis of cis -regulatory elements involved in the activation of IL-6 promoter regulated by N protein. (A) Diagram of site-specific mutants of IL-6 promoter (left panel) and effects of N protein on the activities of these promoters (right panel). (B) Diagram of truncated mutants of IL-6 promoter (left panel) and effects of N protein on the activities of these promoters (right panel). A549 cells were co-transfected with plasmid pCMV-Tag2B-N and reporter plasmid carrying the luciferase reporter gene driven by each of the IL-6 mutant promoters. Promoter activities were determined by measuring the relative luciferase activity in transfected cells. pCMV-Tag2B was used as a negative control (1-fold). Data are expressed as mean + S.D. of three independent experiments.

    Journal: Virology

    Article Title: Nucleocapsid protein of SARS-CoV activates interleukin-6 expression through cellular transcription factor NF-κB

    doi: 10.1016/j.virol.2007.04.009

    Figure Lengend Snippet: Functional analysis of cis -regulatory elements involved in the activation of IL-6 promoter regulated by N protein. (A) Diagram of site-specific mutants of IL-6 promoter (left panel) and effects of N protein on the activities of these promoters (right panel). (B) Diagram of truncated mutants of IL-6 promoter (left panel) and effects of N protein on the activities of these promoters (right panel). A549 cells were co-transfected with plasmid pCMV-Tag2B-N and reporter plasmid carrying the luciferase reporter gene driven by each of the IL-6 mutant promoters. Promoter activities were determined by measuring the relative luciferase activity in transfected cells. pCMV-Tag2B was used as a negative control (1-fold). Data are expressed as mean + S.D. of three independent experiments.

    Article Snippet: All mutant N genes were cloned into Eco RI and Bam HI sites of pCMV-tag2B (Stratagene) to generate plasmids pCMV-tag2B-NΔ220-231, pCMV-tag2B-N1-422, pCMV-tag2B-N1-341, pCMV-tag2B-N61-422S, pCMV-tag2B-N86-422, pCMV-tag2B-N96-42, and pCMV-tag2B-N86-341, respectively.

    Techniques: Functional Assay, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Mutagenesis, Activity Assay, Negative Control

    Determination of the role of N protein in the translocation of NF-κB and activation of IL-6. (A) A549 cells were transfected with plasmid pCMV-Tag2B-N. Cytosol and nucleus fractions of transfected cells were prepared at different time as indicated. Levels of cytosolic and nuclear p65 protein and N protein were determined by western blot using anti-p65 antibody and anti-N antibody, respectively. (B) A549 cells were transfected with plasmid pCMV-Tag2B-M. Cytosol and nucleus fractions of transfected cells were prepared at different time as indicated. Levels of cytosolic and nuclear p65 protein were determined by western blot using anti-p65 antibody. (C) A549 cells were co-transfected with reporter plasmid pIL6-luc-651 along with plasmid pCMV-Tag2B-N or plasmid pCMV-Tag2B-MutNΔ220-231 expressing a mutant N protein (ΔNES) in which a putative nuclear export signal NES located at position 220–231 was deleted. IL-6 promoter activities were determined by measuring the relative luciferase activity in transfected cells. Data are expressed as mean + S.D. of three independent experiments.

    Journal: Virology

    Article Title: Nucleocapsid protein of SARS-CoV activates interleukin-6 expression through cellular transcription factor NF-κB

    doi: 10.1016/j.virol.2007.04.009

    Figure Lengend Snippet: Determination of the role of N protein in the translocation of NF-κB and activation of IL-6. (A) A549 cells were transfected with plasmid pCMV-Tag2B-N. Cytosol and nucleus fractions of transfected cells were prepared at different time as indicated. Levels of cytosolic and nuclear p65 protein and N protein were determined by western blot using anti-p65 antibody and anti-N antibody, respectively. (B) A549 cells were transfected with plasmid pCMV-Tag2B-M. Cytosol and nucleus fractions of transfected cells were prepared at different time as indicated. Levels of cytosolic and nuclear p65 protein were determined by western blot using anti-p65 antibody. (C) A549 cells were co-transfected with reporter plasmid pIL6-luc-651 along with plasmid pCMV-Tag2B-N or plasmid pCMV-Tag2B-MutNΔ220-231 expressing a mutant N protein (ΔNES) in which a putative nuclear export signal NES located at position 220–231 was deleted. IL-6 promoter activities were determined by measuring the relative luciferase activity in transfected cells. Data are expressed as mean + S.D. of three independent experiments.

    Article Snippet: All mutant N genes were cloned into Eco RI and Bam HI sites of pCMV-tag2B (Stratagene) to generate plasmids pCMV-tag2B-NΔ220-231, pCMV-tag2B-N1-422, pCMV-tag2B-N1-341, pCMV-tag2B-N61-422S, pCMV-tag2B-N86-422, pCMV-tag2B-N96-42, and pCMV-tag2B-N86-341, respectively.

    Techniques: Translocation Assay, Activation Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Mutagenesis, Luciferase, Activity Assay

    Determination of the role of NF-κB in the expression of IL-6 regulated by N protein. (A) A549 cells were co-transfected with pCMV-Tag2B-N and pIL6-luc-651 followed by the treatment with MG-132 at different concentrations as indicated for 24h. Treated cells were then lysed and luciferase activity was measured. (B) A549 cells were co-transfected with pIL6-luc-651 (lanes 1–4) or pIL6-luc-651ΔNF-κB (lanes 5, 6, 7, and 8), pCMV-Tag2B-N (lanes 2, 3, 6, and 7) or pCMV-Tag2B (lanes 1, 2, 4–6, and 8), and pCMV-Tag2B-p65 (lanes 3, 4, 7, and 8), respectively. Transfected cells were then lysed and luciferase activity was measured. (C) A549 cells were transfected with pCMV-Tag2B (lanes 2 and 3), or pCMV-Tag2B-N (lanes 3 and 4), or pCMV-Tag2B-p65 (lanes 4 and 5). Transfected cells were lysed and the levels of IL-6 protein produced were then measured by ELISA using anti-IL-6 antibody. The results were expressed as mean + S.D. of three independent experiments.

    Journal: Virology

    Article Title: Nucleocapsid protein of SARS-CoV activates interleukin-6 expression through cellular transcription factor NF-κB

    doi: 10.1016/j.virol.2007.04.009

    Figure Lengend Snippet: Determination of the role of NF-κB in the expression of IL-6 regulated by N protein. (A) A549 cells were co-transfected with pCMV-Tag2B-N and pIL6-luc-651 followed by the treatment with MG-132 at different concentrations as indicated for 24h. Treated cells were then lysed and luciferase activity was measured. (B) A549 cells were co-transfected with pIL6-luc-651 (lanes 1–4) or pIL6-luc-651ΔNF-κB (lanes 5, 6, 7, and 8), pCMV-Tag2B-N (lanes 2, 3, 6, and 7) or pCMV-Tag2B (lanes 1, 2, 4–6, and 8), and pCMV-Tag2B-p65 (lanes 3, 4, 7, and 8), respectively. Transfected cells were then lysed and luciferase activity was measured. (C) A549 cells were transfected with pCMV-Tag2B (lanes 2 and 3), or pCMV-Tag2B-N (lanes 3 and 4), or pCMV-Tag2B-p65 (lanes 4 and 5). Transfected cells were lysed and the levels of IL-6 protein produced were then measured by ELISA using anti-IL-6 antibody. The results were expressed as mean + S.D. of three independent experiments.

    Article Snippet: All mutant N genes were cloned into Eco RI and Bam HI sites of pCMV-tag2B (Stratagene) to generate plasmids pCMV-tag2B-NΔ220-231, pCMV-tag2B-N1-422, pCMV-tag2B-N1-341, pCMV-tag2B-N61-422S, pCMV-tag2B-N86-422, pCMV-tag2B-N96-42, and pCMV-tag2B-N86-341, respectively.

    Techniques: Expressing, Transfection, Luciferase, Activity Assay, Produced, Enzyme-linked Immunosorbent Assay

    Interaction of jTat with CycT1 in vitro and in vivo . (A) Interaction of hTat and jTat with mammalian CycT1s. GST and GST-tagged proteins were immobilized on beads and incubated with the cell lysates as described in Methods. The pull-down complexes and 5% of cell lysate input were analyzed by western-blotting using anti-Flag antibody. The coomassie blue staining shows 10% of the amounts of the purified proteins utilized in this experiment. Numbers mark the molecular weight standards (MW). (B) Schematic representation of mammalian two-hybrid constructs. jAD; jTat residues 1-67. JH; jTat 1-67 fused to hTat 48-72. (C) HeLa cells were co-transfected with 500 ng J-NFκB or H-NFκB, 500 ng of the indicated Gal4 BD plasmid and 250 ng pFR- luc . Fold-induction shows the relative activity of pFR- luc reporter and reflects binding affinity between Tat and its cofactor. (D) HIV LTR activation in 3T3 cells by indicated Tats in the absence or presence of hCycT1. The amount of transfected pCMV-Tag2B-hCycT1 was 50 ng. T1; Cyclin T1 residues 1-272.

    Journal: Virology Journal

    Article Title: Comparative functional analysis of Jembrana disease virus Tat protein on lentivirus long terminal repeat promoters: evidence for flexibility at its N-terminus

    doi: 10.1186/1743-422X-6-179

    Figure Lengend Snippet: Interaction of jTat with CycT1 in vitro and in vivo . (A) Interaction of hTat and jTat with mammalian CycT1s. GST and GST-tagged proteins were immobilized on beads and incubated with the cell lysates as described in Methods. The pull-down complexes and 5% of cell lysate input were analyzed by western-blotting using anti-Flag antibody. The coomassie blue staining shows 10% of the amounts of the purified proteins utilized in this experiment. Numbers mark the molecular weight standards (MW). (B) Schematic representation of mammalian two-hybrid constructs. jAD; jTat residues 1-67. JH; jTat 1-67 fused to hTat 48-72. (C) HeLa cells were co-transfected with 500 ng J-NFκB or H-NFκB, 500 ng of the indicated Gal4 BD plasmid and 250 ng pFR- luc . Fold-induction shows the relative activity of pFR- luc reporter and reflects binding affinity between Tat and its cofactor. (D) HIV LTR activation in 3T3 cells by indicated Tats in the absence or presence of hCycT1. The amount of transfected pCMV-Tag2B-hCycT1 was 50 ng. T1; Cyclin T1 residues 1-272.

    Article Snippet: The full-length CDK9, human cyclin T2 isoform B (CycT2b) and residues 1-272 of human, bovine and murine CycT1, were kind gifts from Alan Frankel (University of California, San Francisco) and subcloned to pcDNA3.1 (+) and pCMV-Tag2B vectors (Stratagene).

    Techniques: In Vitro, In Vivo, Incubation, Western Blot, Staining, Purification, Molecular Weight, Construct, Transfection, Plasmid Preparation, Activity Assay, Binding Assay, Activation Assay

    Function of different Danio rerio DDX41 ( Dr DDX41) domains in the signaling pathways. (A) Schematic diagram of full-length and mutated Dr DDX41 fragments, which were inserted into the C-terminus of pCMV-Tag2B. (B,C) Dr DDX41 (B) and its mutants [ (C) , constructed as in panel (A) ] interact with Danio rerio STING ( Dr STING). HEK293T cells were transfected with Flag- Dr DDX41/its mutants and Myc- Dr STING for 24 h. Cell lysates were immunoprecipitated with anti-Myc antibody (Myc) and analyzed by western blot using anti-Flag and anti-Myc antibodies. Expression of the transfected plasmids was analyzed with anti-Flag and anti-Myc antibodies in the whole cell lysates. (D–F) Activation of the nuclear factor-κB (NF-κB)-binding (D) , Hs IRF3 (E) , or Hs IFNβ (F) promoters in HEK293T cells transfected with an NF-κB/ Hs IRF3/ Hs IFNβ-Luc reporter (150 ng/mL) and a renilla luciferase reporter (15 ng/mL) plus different vectors of Dr DDX41 (400 ng/mL) together with pCMV-N-Myc- Dr STING (200 ng/mL) and stimulated with poly(dA:dT) (1 µg/mL) for 6 h. Data are the average luciferase activity ± SD (* p

    Journal: Frontiers in Immunology

    Article Title: Identification of DEAD-Box RNA Helicase DDX41 as a Trafficking Protein That Involves in Multiple Innate Immune Signaling Pathways in a Zebrafish Model

    doi: 10.3389/fimmu.2018.01327

    Figure Lengend Snippet: Function of different Danio rerio DDX41 ( Dr DDX41) domains in the signaling pathways. (A) Schematic diagram of full-length and mutated Dr DDX41 fragments, which were inserted into the C-terminus of pCMV-Tag2B. (B,C) Dr DDX41 (B) and its mutants [ (C) , constructed as in panel (A) ] interact with Danio rerio STING ( Dr STING). HEK293T cells were transfected with Flag- Dr DDX41/its mutants and Myc- Dr STING for 24 h. Cell lysates were immunoprecipitated with anti-Myc antibody (Myc) and analyzed by western blot using anti-Flag and anti-Myc antibodies. Expression of the transfected plasmids was analyzed with anti-Flag and anti-Myc antibodies in the whole cell lysates. (D–F) Activation of the nuclear factor-κB (NF-κB)-binding (D) , Hs IRF3 (E) , or Hs IFNβ (F) promoters in HEK293T cells transfected with an NF-κB/ Hs IRF3/ Hs IFNβ-Luc reporter (150 ng/mL) and a renilla luciferase reporter (15 ng/mL) plus different vectors of Dr DDX41 (400 ng/mL) together with pCMV-N-Myc- Dr STING (200 ng/mL) and stimulated with poly(dA:dT) (1 µg/mL) for 6 h. Data are the average luciferase activity ± SD (* p

    Article Snippet: Plasmid Construction For functional analysis, the coding sequences (CDSs) for the full-length and various domain/residue-deleted mutant Dr DDX41 proteins were inserted into pCMV-Tag2B (Invitrogen) between BamHI/XhoI sites to construct eukaryotic expression vectors, including pCMV-Tag2B-Dr DDX41 (full-length), pCMV-Tag2B-Dr DDX41-ΔN (177–613 amino acids, lacking N-terminal region), pCMV-Tag2B-Dr DDX41-(ΔN + ΔDEADc) (412–613 amino acids, lacking N-terminal region and DEADc domain), pCMV-Tag2B-Dr DDX41-(ΔC + ΔHELICc) (1–411 amino acids, lacking C-terminal region and HELICc domain), and pCMV-Tag2B-Dr DDX41-ΔC (1–558 amino acids, lacking C-terminal region).

    Techniques: Construct, Transfection, Immunoprecipitation, Western Blot, Expressing, Activation Assay, Binding Assay, Luciferase, Activity Assay

    Ectopic EDD increases cisplatin/doxorubicin resistance in MDA-MB-436 cells. (A, B) MDA-MB-436 cells were transfected with increasing concentrations of plasmid pCMV-Tag2B or pCMV-Tag2B.EDD or left untransfected (Con). After 2 days, the cells were harvested for semi-quantitative RT-PCR (A, B; upper panel ) or Western (B; lower panel ) analyses to confirm elevated EDD expression. Each is a representative of 4 experiments. (C, F) MDA-MB-436 cells were treated with increasing doses of cisplatin (C) or doxorubicin (F) for 5 days and cells were counted to determine IC 50 values. Mean ± SEM from 2 independent experiments, each in triplicate. (D, E, G, H) MDA-MB-436 cells were transfected with pCMV-Tag2B or pCMV-Tag2B.EDD. After 24 h (Day 1), the cells were treated ± IC 50 doses of (D, E) cisplatin at 0.3125 μM or (G, H) doxorubicin at 0.023 μM. On Day 5, cells were counted using trypan-blue assay. The results were plotted as (D, G) cell numbers ± drug treatment or (E, H) % cell survival of drug-treated pCMV-Tag2B or pCMV-Tag2B.EDD transfectants, each compared to its untreated control. Mean ± SEM (n = 4-5), *P

    Journal: American Journal of Cancer Research

    Article Title: Prolactin-inducible EDD E3 ubiquitin ligase promotes TORC1 signalling, anti-apoptotic protein expression, and drug resistance in breast cancer cells

    doi:

    Figure Lengend Snippet: Ectopic EDD increases cisplatin/doxorubicin resistance in MDA-MB-436 cells. (A, B) MDA-MB-436 cells were transfected with increasing concentrations of plasmid pCMV-Tag2B or pCMV-Tag2B.EDD or left untransfected (Con). After 2 days, the cells were harvested for semi-quantitative RT-PCR (A, B; upper panel ) or Western (B; lower panel ) analyses to confirm elevated EDD expression. Each is a representative of 4 experiments. (C, F) MDA-MB-436 cells were treated with increasing doses of cisplatin (C) or doxorubicin (F) for 5 days and cells were counted to determine IC 50 values. Mean ± SEM from 2 independent experiments, each in triplicate. (D, E, G, H) MDA-MB-436 cells were transfected with pCMV-Tag2B or pCMV-Tag2B.EDD. After 24 h (Day 1), the cells were treated ± IC 50 doses of (D, E) cisplatin at 0.3125 μM or (G, H) doxorubicin at 0.023 μM. On Day 5, cells were counted using trypan-blue assay. The results were plotted as (D, G) cell numbers ± drug treatment or (E, H) % cell survival of drug-treated pCMV-Tag2B or pCMV-Tag2B.EDD transfectants, each compared to its untreated control. Mean ± SEM (n = 4-5), *P

    Article Snippet: Cells receiving pCMV-Tag2B or pCMV-Tag2B.EDD above 250 ng/ml, grown in drug-free conditions, generally showed a progressive decrease in cell counts as compared to untransfected cells ( , ).

    Techniques: Multiple Displacement Amplification, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Expressing

    Overexpression of KLF2 expression inhibits HepG2 cell proliferation and improves apoptosis. (A) The mRNA level of KLF2 in HepG2 and Hep3B cells transfected with pCMV-Tag2B-KLF2 or empty vector was detected by qPCR. (B,C) MTT assays and colony formation assays were used to determine the cell viability for pCMV-Tag2B-KLF2-transfected or empty vector-transfected HepG2 and Hep3B cells. Values represent the mean ± s.d. from three independent experiments. (D) Apoptosis was determined by flow cytometry. UL, necrotic cells; UR, terminal apoptotic cells; LR, early apoptotic cells. * P

    Journal: Journal of Hematology & Oncology

    Article Title: Long non-coding RNA ANRIL is upregulated in hepatocellular carcinoma and regulates cell apoptosis by epigenetic silencing of KLF2

    doi: 10.1186/s13045-015-0146-0

    Figure Lengend Snippet: Overexpression of KLF2 expression inhibits HepG2 cell proliferation and improves apoptosis. (A) The mRNA level of KLF2 in HepG2 and Hep3B cells transfected with pCMV-Tag2B-KLF2 or empty vector was detected by qPCR. (B,C) MTT assays and colony formation assays were used to determine the cell viability for pCMV-Tag2B-KLF2-transfected or empty vector-transfected HepG2 and Hep3B cells. Values represent the mean ± s.d. from three independent experiments. (D) Apoptosis was determined by flow cytometry. UL, necrotic cells; UR, terminal apoptotic cells; LR, early apoptotic cells. * P

    Article Snippet: Overexpression of KLF2 impairs HCC cell proliferation and induces cell apoptosis To determine whether KLF2 is involved in ANRIL-mediated increase in HCC cell proliferation, we upregulated KLF2 expression in HCC cells by transfecting with a FLAG-tagged KLF2 expression vector using the pCMV-Tag2B vector (Stratagene, Santa Clara, CA, USA).

    Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, MTT Assay, Flow Cytometry, Cytometry

    The MDM2 3-11-12 s minigene undergoes damage-induced exon 11 skipping in an in vitro splicing system while a control p53 7-8-9 minigene remains unresponsive. ( A ) A minimal MDM2 3-11-12 s minigene, constructed to assess the elements essential for the generation of MDM2-ALT1 alternative splicing, was derived from the previously described MDM2 3-11-12 minigene, which is responsive to stress-induced alternative splicing. The schematic represents the 3-11-12 s minigene and the sizes depicted reflect the length of the exonic and intronic regions of the minigene construct and are inclusive of the Flag-tag and the intervening region (cloning sites) of the pCMV-tag2B vector at the 5′ end of the minigene construct. In vitro -transcribed RNA obtained from the minigenes was subjected to a cell-free in vitro splicing assay using nuclear extracts from either normal (N, NOR) or cisplatinum-treated HeLa S3 cells (C, CIS). RNA was isolated, reversed transcribed and subjected to a 25-cycle PCR using γ- 32 P-radioactively-labeled Flag primer and gene-specific reverse primers. The MDM2 minigene predominantly skips internal exon 11 when spliced in nuclear extracts from cisplatinum-treated cells, but not in nuclear extract from normal cells. The bar graphs represent the percentage of 3.12 skipped product obtained from three independent in vitro splicing experiments under each condition and the error bars represent standard error mean (SEM). The difference in the percentage of 3.12 product between normal and damaged splicing conditions is statistically significant ( n = 3). *Indicates non-specific band also seen in –ATP controls (see Supplementary Figure S1). ∧ Indicates probable PCR degradation products. ( B ) Damage-responsive alternative splicing is transcript-specific. A p53 7-8-9 minigene shows no changes in splicing patterns between the normal and damaged nuclear extract ( n = 3). The sizes of the minigene depicted in the schematic are reflective of the Flag-tag and vector-specific regions at the 5′ end of the minigene construct in a manner similar to the 3-11-12 s minigene.

    Journal: Nucleic Acids Research

    Article Title: Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage

    doi: 10.1093/nar/gkv223

    Figure Lengend Snippet: The MDM2 3-11-12 s minigene undergoes damage-induced exon 11 skipping in an in vitro splicing system while a control p53 7-8-9 minigene remains unresponsive. ( A ) A minimal MDM2 3-11-12 s minigene, constructed to assess the elements essential for the generation of MDM2-ALT1 alternative splicing, was derived from the previously described MDM2 3-11-12 minigene, which is responsive to stress-induced alternative splicing. The schematic represents the 3-11-12 s minigene and the sizes depicted reflect the length of the exonic and intronic regions of the minigene construct and are inclusive of the Flag-tag and the intervening region (cloning sites) of the pCMV-tag2B vector at the 5′ end of the minigene construct. In vitro -transcribed RNA obtained from the minigenes was subjected to a cell-free in vitro splicing assay using nuclear extracts from either normal (N, NOR) or cisplatinum-treated HeLa S3 cells (C, CIS). RNA was isolated, reversed transcribed and subjected to a 25-cycle PCR using γ- 32 P-radioactively-labeled Flag primer and gene-specific reverse primers. The MDM2 minigene predominantly skips internal exon 11 when spliced in nuclear extracts from cisplatinum-treated cells, but not in nuclear extract from normal cells. The bar graphs represent the percentage of 3.12 skipped product obtained from three independent in vitro splicing experiments under each condition and the error bars represent standard error mean (SEM). The difference in the percentage of 3.12 product between normal and damaged splicing conditions is statistically significant ( n = 3). *Indicates non-specific band also seen in –ATP controls (see Supplementary Figure S1). ∧ Indicates probable PCR degradation products. ( B ) Damage-responsive alternative splicing is transcript-specific. A p53 7-8-9 minigene shows no changes in splicing patterns between the normal and damaged nuclear extract ( n = 3). The sizes of the minigene depicted in the schematic are reflective of the Flag-tag and vector-specific regions at the 5′ end of the minigene construct in a manner similar to the 3-11-12 s minigene.

    Article Snippet: Following this, the inserts were ligated into the pCMV-Tag2B vector, digested with BamHI and HindIII, and then transformed into stellar competent cells according the manufacturer's protocols.

    Techniques: In Vitro, Construct, Derivative Assay, FLAG-tag, Clone Assay, Plasmid Preparation, Splicing Assay, Isolation, Polymerase Chain Reaction, Labeling

    The interaction with H19 favors the decay-promoting function of KSRP. ( A ) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-KSRP (flag-KSRP), or pTAG2B-H19 (H19) plus pCDNA3-Flag-KSRP (flag-KSRP). Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. ( B ) HEK-293 cells were transiently transfected with GST-fused MS2 binding protein (GST-MS2BP) together with pTAG2B-myog 3′UTR and pTAG2B-MS2-12XH19 (in which murine H19 was tagged with MS2 RNA hairpins; MS2-H19) or pTAG2B-MS2-12X (empty vector; MS2). Total cell extracts were prepared 48 h after transfection and precipitated by glutathione–Sepharose beads. RNA was purified and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. ( C ) HEK-293 cells were transiently cotransfected with pCMV-TAG2B-KSRP (expressing Flag-tagged WT KSRP) or pCMV-TAG2B expressing the indicated Flag-tagged KSRP mutants (KH1GDDG, KH2GDDG, KH3GDDG, KH4GDDG) together with pTAG2B-myog 3′UTR and pTAG2B-H19. Total cell extracts were prepared 48 h after transfection and immunoprecipitated by anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect myogenin 3′UTR (m.Myog 3′UTR) or transfected murine H19 (m.H19). ( D ) In vitro RNA degradation assays using S100 extracts from shKSRP C2C12 cells preincubated (for 90 min at 4 °C) with anti-Flag immunoprecipitates from HEK-293 cells transiently transfected with Flag-tagged WT KSRP (KSRP) or Flag-tagged KH1GDDG mutant (KSRP[KH1GDDG]) together with the E3 sequence or murine H19 cloned in expression vectors. Internally 32 P-labeled and capped RNA substrates were incubated with the aforementioned reaction mixtures, and their decay was monitored for the indicated times. RNA was analyzed by denaturing polyacrylamide gel electrophoresis followed by autoradiography. E3 is a stable transcript used to detect background decay. Representative autoradiograms are displayed. ( E ) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5, or pTAG2B-H19 (H19) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5. Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. The values of RT-qPCR experiments shown are averages (±SEM) of three independent experiments performed in triplicate (* P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: H19 long noncoding RNA controls the mRNA decay promoting function of KSRP

    doi: 10.1073/pnas.1415098111

    Figure Lengend Snippet: The interaction with H19 favors the decay-promoting function of KSRP. ( A ) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-KSRP (flag-KSRP), or pTAG2B-H19 (H19) plus pCDNA3-Flag-KSRP (flag-KSRP). Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. ( B ) HEK-293 cells were transiently transfected with GST-fused MS2 binding protein (GST-MS2BP) together with pTAG2B-myog 3′UTR and pTAG2B-MS2-12XH19 (in which murine H19 was tagged with MS2 RNA hairpins; MS2-H19) or pTAG2B-MS2-12X (empty vector; MS2). Total cell extracts were prepared 48 h after transfection and precipitated by glutathione–Sepharose beads. RNA was purified and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. ( C ) HEK-293 cells were transiently cotransfected with pCMV-TAG2B-KSRP (expressing Flag-tagged WT KSRP) or pCMV-TAG2B expressing the indicated Flag-tagged KSRP mutants (KH1GDDG, KH2GDDG, KH3GDDG, KH4GDDG) together with pTAG2B-myog 3′UTR and pTAG2B-H19. Total cell extracts were prepared 48 h after transfection and immunoprecipitated by anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect myogenin 3′UTR (m.Myog 3′UTR) or transfected murine H19 (m.H19). ( D ) In vitro RNA degradation assays using S100 extracts from shKSRP C2C12 cells preincubated (for 90 min at 4 °C) with anti-Flag immunoprecipitates from HEK-293 cells transiently transfected with Flag-tagged WT KSRP (KSRP) or Flag-tagged KH1GDDG mutant (KSRP[KH1GDDG]) together with the E3 sequence or murine H19 cloned in expression vectors. Internally 32 P-labeled and capped RNA substrates were incubated with the aforementioned reaction mixtures, and their decay was monitored for the indicated times. RNA was analyzed by denaturing polyacrylamide gel electrophoresis followed by autoradiography. E3 is a stable transcript used to detect background decay. Representative autoradiograms are displayed. ( E ) HEK-293 cells were transiently cotransfected with pTAG2B-myog 3′UTR together with empty vector (mock control cells), or pTAG2B-E3 (E3) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5, or pTAG2B-H19 (H19) plus pCDNA3-Flag-EXOSC2 and pCDNA3-Flag-EXOSC5. Total cell extracts were prepared 48 h after transfection and immunoprecipitated with anti-Flag antibody. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect transfected murine myogenin 3′UTR (m.Myog 3′UTR) or endogenous GNAS. The values of RT-qPCR experiments shown are averages (±SEM) of three independent experiments performed in triplicate (* P

    Article Snippet: To generate pTAG2B-H19 expression vector, a fragment of 2,166 nt from murine H19 obtained by RT-qPCR was cloned in the EcoRI/SalI sites of pCMV-TAG2B plasmid (Agilent).

    Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Purification, Quantitative RT-PCR, Binding Assay, Expressing, In Vitro, Mutagenesis, Sequencing, Clone Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography