transient transfections Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore pei transfection transient transfections
    Pei Transfection Transient Transfections, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pei transfection transient transfections/product/Millipore
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    pei transfection transient transfections - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    93
    OriGene transient transfection
    Site-directed mutagenesis for luciferase activity assay and effect of miR-7 silencing over MAFG expression. (A) Chromosomal localization of miR-7 predicted binding sites at 3'UTR of MAFG. Regions 2 and 8 were identified by six or more bioinformatical algorithms. Sanger sequencing showed that the seed sequence of miR-7 was fully mutated at regions 2 and 8 of the 3' UTR of MAFG. (B) <t>Co-transfection</t> of mimic miR-7 (miR-7) or mimic control (miR-NC) with the 3' UTR of MAFG WT, mutated on region 2 (MAFG 2*) and mutated on region 8 (MAFG 8*). Experiments were performed at 15nM and data was analyzed after 24h of co-transfection. (Upper panel) Relative luciferase activity. The figures represent the mean ± SD of at least 3 independent experiments after data normalization with Renilla and the data from the negative control 3'-UTR; p
    Transient Transfection, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 607 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection/product/OriGene
    Average 93 stars, based on 607 article reviews
    Price from $9.99 to $1999.99
    transient transfection - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    94
    Roche transient transfection
    Mapping of the RNA sequence(s) in HBV pregenomic RNA responsible for HBV RNA decay. (A) Schematic diagram of the truncation and deletion mutants of HBV pregenomic RNA. The numbers indicate the HBV DNA sequence with 1 at the unique EcoRI site in the HBV genome. (B) Huh7 cells were transfected with 0.1 μg of luciferase fusion constructs containing the truncation mutants of HBV DNA and 0.2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h, and the luciferase activity was then assessed. For each <t>transfection,</t> 0.01 μg of pRL-tk was included as an internal control of transfection efficiency. Results represent the means of data from three independent experiments performed in duplicate. *, P
    Transient Transfection, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 5743 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection/product/Roche
    Average 94 stars, based on 5743 article reviews
    Price from $9.99 to $1999.99
    transient transfection - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    93
    Thermo Fisher transient transfection
    Relative change in TAG- or CE-specific intracellular lipid storage upon expression of various Plin proteins. AML12 cells were transiently transfected with the indicated GFP–Plin-expressing constructs and cultured overnight in the presence of oleic acid and cholesterol. <t>Transfection</t> efficiencies were confirmed by visualizing GFP fluorescence. Untransfected cells, cultured with/without exogenous oleic acid and cholesterol, were grown in parallel. LSDs were isolated by centrifugation and lipids were extracted, separated by TLC in parallel with lipid markers, and TAG and CE detected after staining with iodine vapor. Relative TAG/CE ratios were quantified in cells transfected with each specific Plin-expressing construct and analyzed in parallel with identically grown untransfected cells for normalization and TLC background correction. The TAG/CE ratio for untransfected lipid-loaded cells (controls) was set to 0.5. The relative TAG/CE-ratio of Plin1a- and Plin1c-expressing cells were always analyzed in parallel and normalized to those of control cells, and then secondarily compared to results determined for its Plin-expressing counterpart. Values > 0.5 indicate a proportional increase in TAG lipid bias, whereas values
    Transient Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 30308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection/product/Thermo Fisher
    Average 93 stars, based on 30308 article reviews
    Price from $9.99 to $1999.99
    transient transfection - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    93
    Horizon Discovery transient transfection
    Outline of pharmacological manipulation of CK1 functions and its effects on p53 signalling pathway, cell viability, and CK1α post-translational modifications. ( A ) H1299 cells were transfected with STReP-tagged CK1α sp1 or 2, FLAG-tagged peptide 35 and His-Ubiquitin or His-Nedd8, using Attractene. Eighteen hours after <t>transfection</t> cells were treated with 50 µM MG132 for 4 hours. Whole cell lysates were analysed for transfected CK1α levels, and His-ubiquitin/NEDD8 conjugates were purified by metal affinity chromatography and analyzed by 4%–12% NuPAGE/immunoblots with an anti-CK1α antibody. Changes highlighted were representative of two independent experiments. ( B ) Three approaches for manipulation of signal transduction pathways. Genetic depletion of CK1α using siRNA or the CK1 kinase attenuation using the ATP-competitive inhibitor D4476 can alter the p53 response and cell viability, as indicated. The bioactive peptide from the high affinity MDM2-CK1α interface (peptide 35) highlights a novel peptide lead for disrupting signalling in cancers. All three approaches gave rise to overlapping but distinct effects on signalling. Specific siRNA against CK1α led to p53 induction and growth arrest accompanied with slight sub-G1 increase, whereas non-specific CK1 drugs such as D4476 mediated p53 induction and significant apoptosis. Low levels of the bioactive peptide 35 did not lead to p53 protein induction but to a growth arrest in G0-G1 and reduced cell viability. These data suggest that peptide 35 may function in a pharmacologically novel manner compared to the ATP-active site inhibitor or CK1α siRNA and highlights the general utility of targeting protein-protein interactions as approaches for developing therapeutic strategies that target signalling mechanisms in cancer.
    Transient Transfection, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 93/100, based on 662 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection/product/Horizon Discovery
    Average 93 stars, based on 662 article reviews
    Price from $9.99 to $1999.99
    transient transfection - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    94
    Promega transient transfection
    Stimulation-induced processing and degradation of p105 is not affected by mutating lysine residues 18 (683) to 30 (967) in the C-terminal domain of the molecule. (A) COS-7 cells were transiently transfected with either a cDNA coding for p105-WT (lanes 2 to 5) or p105-K18-30R (lanes 6 to 9) as indicated. Control cells (lane 1) were transfected with an empty vector. Where indicated, a cDNA coding for constitutively active IKKβ was cotransfected. Twenty-four hours after <t>transfection</t> cells were harvested, and nuclear and cytosolic fractions were isolated as described in Materials and Methods. Aliquots of cytosolic and nuclear extracts representing an equal number of cells were resolved via SDS-10% PAGE and were blotted onto nitrocellulose paper, and proteins were visualized by using anti-p50 antibody and ECL as described in Materials and Methods. C, cytosolic fraction; N, nuclear fraction. (B) COS-7 cells were transiently transfected with a cDNA coding for either p105-WT (lanes 1 to 4), p105-K18-30R (lanes 5 to 8), or p105-Δ917-932 (lanes 9 to 12). Where indicated, cDNA coding for a constitutively active IKKβ was cotransfected. Twenty-four hours after transfection cells were pulse labeled with [ 35 S]methionine (0; pulse). Following removal and dilution of the labeled amino acid and further incubation for 2 h (2; chase), the labeled proteins were immunoprecipitated by using anti-p50 antibody, resolved by SDS-10% PAGE, and visualized by phosphorimaging as described in Materials and Methods.
    Transient Transfection, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 3476 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection/product/Promega
    Average 94 stars, based on 3476 article reviews
    Price from $9.99 to $1999.99
    transient transfection - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    91
    Cisbio Bioassays transient transfection
    Stimulation-induced processing and degradation of p105 is not affected by mutating lysine residues 18 (683) to 30 (967) in the C-terminal domain of the molecule. (A) COS-7 cells were transiently transfected with either a cDNA coding for p105-WT (lanes 2 to 5) or p105-K18-30R (lanes 6 to 9) as indicated. Control cells (lane 1) were transfected with an empty vector. Where indicated, a cDNA coding for constitutively active IKKβ was cotransfected. Twenty-four hours after <t>transfection</t> cells were harvested, and nuclear and cytosolic fractions were isolated as described in Materials and Methods. Aliquots of cytosolic and nuclear extracts representing an equal number of cells were resolved via SDS-10% PAGE and were blotted onto nitrocellulose paper, and proteins were visualized by using anti-p50 antibody and ECL as described in Materials and Methods. C, cytosolic fraction; N, nuclear fraction. (B) COS-7 cells were transiently transfected with a cDNA coding for either p105-WT (lanes 1 to 4), p105-K18-30R (lanes 5 to 8), or p105-Δ917-932 (lanes 9 to 12). Where indicated, cDNA coding for a constitutively active IKKβ was cotransfected. Twenty-four hours after transfection cells were pulse labeled with [ 35 S]methionine (0; pulse). Following removal and dilution of the labeled amino acid and further incubation for 2 h (2; chase), the labeled proteins were immunoprecipitated by using anti-p50 antibody, resolved by SDS-10% PAGE, and visualized by phosphorimaging as described in Materials and Methods.
    Transient Transfection, supplied by Cisbio Bioassays, 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/transient transfection/product/Cisbio Bioassays
    Average 91 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    transient transfection - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    92
    Amaxa transient transfection
    YFP-N300 downregulates MHC class I expression in M553(R240)tpsn cells M553(R240)Tpsn cells were transiently transfected with YFP-N300 or co-transfected with empty vector and pmaxGFP. Twenty-four hours post <t>transfection,</t> MHC class I expression was analyzed by flow cytometry using a PE directly conjugated W6/32 (MHC class I) antibody. Mean fluorescent intensity was determined for W6/32 staining on populations of cells gated on forward and side scatter (R1) and for the negative or positive expression of GFP/YFP (R2, GFP/YFP negative) and R3, (GFP/YFP positive).
    Transient Transfection, supplied by Amaxa, used in various techniques. Bioz Stars score: 92/100, based on 995 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection/product/Amaxa
    Average 92 stars, based on 995 article reviews
    Price from $9.99 to $1999.99
    transient transfection - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    Valiant transient transfection
    YFP-N300 downregulates MHC class I expression in M553(R240)tpsn cells M553(R240)Tpsn cells were transiently transfected with YFP-N300 or co-transfected with empty vector and pmaxGFP. Twenty-four hours post <t>transfection,</t> MHC class I expression was analyzed by flow cytometry using a PE directly conjugated W6/32 (MHC class I) antibody. Mean fluorescent intensity was determined for W6/32 staining on populations of cells gated on forward and side scatter (R1) and for the negative or positive expression of GFP/YFP (R2, GFP/YFP negative) and R3, (GFP/YFP positive).
    Transient Transfection, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection/product/Valiant
    Average 92 stars, based on 51 article reviews
    Price from $9.99 to $1999.99
    transient transfection - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    Santa Cruz Biotechnology transient transfection
    Dose effect of ENZ fragment on BAR efficiency. ( A ) Schematic representation of the Gal4-tagged (gray–blue square) ORF2 fragments. ENZ490Δ fragment was generated from wild-type ORF2 sequence, while Δ348RTCys fragment was generated using codon optimized ORF2 sequence. ( B ) BAR output using different amounts (0.4–0.05 μg) of the wild-type ENZ expression plasmid with 0.4 μg of the RTCys expression plasmid. Empty vector used as filler to ensure equal DNA amounts in <t>transfections.</t> Error bars denote standard deviation ( n = 3). Statistical significance assessed using Student's t -test for paired samples with denoted by *. Representative flasks are shown above corresponding graph bars.
    Transient Transfection, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 666 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection/product/Santa Cruz Biotechnology
    Average 92 stars, based on 666 article reviews
    Price from $9.99 to $1999.99
    transient transfection - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    93
    Mirus Bio transient transfection
    IRE1 activation increases the surface expression of α1(A322D) subunit of GABA A receptors. ( A ) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or XBP1-s (spliced XBP1) plasmids. Forty-eight hrs post <t>transfection,</t> cells were lysed, and total proteins were extracted. The cell lysates are then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B C ) (n = 5 for α1 and n = 3 for BiP, paired t-test). ( D ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface protein expression levels of α1 is shown in ( E ) (n = 5, paired t-test). ( F ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or XBP-s or transfected with XBP-s and treated with lactacystin (2.5 μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( G ) (n = 3, one-way ANOVA followed by Fisher test, *, p
    Transient Transfection, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 93/100, based on 1306 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection/product/Mirus Bio
    Average 93 stars, based on 1306 article reviews
    Price from $9.99 to $1999.99
    transient transfection - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    93
    Stratagene transient transfections
    LMP2A augments activation of the TOPFlash luciferase reporter by β-catenin. (A) 293 cells were transfected with SV-β-galactosidase and either TOPFlash or FOPFlash plus vector, β-catenin, LMP2A, or β-catenin with LMP2A. TOPFlash contains four consensus TCF-4 binding sites in a promoter driving expression of the luciferase gene, and FOPFlash is an analogous construct with the four TCF-4 sites mutated. <t>Transfections</t> and assays were performed in triplicate in two separate experiments, and luciferase activity was normalized to β-galactosidase activity to control for transfection efficiency. The data are presented graphically as TOPFlash activity normalized to FOPFlash activity for each of the indicated conditions. (B) Immunoblot analyses were performed on the lysates used in the luciferase assay described above. Antibodies against HA confirm the presence of transfected HA-tagged LMP2A. Transfected and endogenous β-catenin levels were revealed with an anti-β-catenin antibody, and immunoblot with an antibody against TCF-4 attested to equal levels of this transcription factor in the transfected 293 cells.
    Transient Transfections, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfections/product/Stratagene
    Average 93 stars, based on 182 article reviews
    Price from $9.99 to $1999.99
    transient transfections - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    94
    Polyplus Transfection transient transfection
    Methylation-sensitive binding of CTCF to the first exon of hTERT . (A) In vivo binding of CTCF to the first exon of hTERT in telomerase-positive and negative cell lines was analyzed by ChIP assay using anti-CTCF antibody. PCR coamplification of the test fragments ( hTERT and H19) using as template DNA input fraction and DNA recovered from immunoprecipitated fractions bound by the anti-CTCF antibody. (B) 5′-end-labeled control (F1) and SssI methylase-treated (F1-met) fragments were digested with methylation-sensitive BstUI and analyzed on polyacrylamide gels to verify efficiency of in vitro methylation. ( C ) Control unmethylated (F1) or SssI-methylated (F1-met) fragments were analyzed by gel-shift assay (EMSA). F, free probe; B, CTCF-bound probe. ( D ) Representation of the hTERT sequence cloned into the pTERT-297/ex2/FRT. Arrows represent the localization of the primers used for hTERT methylation analysis of stable transfectant. ( E ) The methylation status of the stable transfectants was verified by MS-SSCA. Unmethylated and fully methylated controls were obtained from plasmids used for stable <t>transfection.</t> UT and MT represent, respectively, the unmethylated and methylated plasmids stably transfected into HeLa cells, and after 30 population doublings. ( F ) Binding of CTCF to the first exon of hTERT in stably transfected cell line was analyzed by ChIP assay using anti-CTCF antibody.
    Transient Transfection, supplied by Polyplus Transfection, used in various techniques. Bioz Stars score: 94/100, based on 1003 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection/product/Polyplus Transfection
    Average 94 stars, based on 1003 article reviews
    Price from $9.99 to $1999.99
    transient transfection - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    93
    Merck & Co transient transfection genejuice
    Methylation-sensitive binding of CTCF to the first exon of hTERT . (A) In vivo binding of CTCF to the first exon of hTERT in telomerase-positive and negative cell lines was analyzed by ChIP assay using anti-CTCF antibody. PCR coamplification of the test fragments ( hTERT and H19) using as template DNA input fraction and DNA recovered from immunoprecipitated fractions bound by the anti-CTCF antibody. (B) 5′-end-labeled control (F1) and SssI methylase-treated (F1-met) fragments were digested with methylation-sensitive BstUI and analyzed on polyacrylamide gels to verify efficiency of in vitro methylation. ( C ) Control unmethylated (F1) or SssI-methylated (F1-met) fragments were analyzed by gel-shift assay (EMSA). F, free probe; B, CTCF-bound probe. ( D ) Representation of the hTERT sequence cloned into the pTERT-297/ex2/FRT. Arrows represent the localization of the primers used for hTERT methylation analysis of stable transfectant. ( E ) The methylation status of the stable transfectants was verified by MS-SSCA. Unmethylated and fully methylated controls were obtained from plasmids used for stable <t>transfection.</t> UT and MT represent, respectively, the unmethylated and methylated plasmids stably transfected into HeLa cells, and after 30 population doublings. ( F ) Binding of CTCF to the first exon of hTERT in stably transfected cell line was analyzed by ChIP assay using anti-CTCF antibody.
    Transient Transfection Genejuice, supplied by Merck & Co, used in various techniques. Bioz Stars score: 93/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection genejuice/product/Merck & Co
    Average 93 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    transient transfection genejuice - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    90
    ScienCell transient transfection hcfs
    Methylation-sensitive binding of CTCF to the first exon of hTERT . (A) In vivo binding of CTCF to the first exon of hTERT in telomerase-positive and negative cell lines was analyzed by ChIP assay using anti-CTCF antibody. PCR coamplification of the test fragments ( hTERT and H19) using as template DNA input fraction and DNA recovered from immunoprecipitated fractions bound by the anti-CTCF antibody. (B) 5′-end-labeled control (F1) and SssI methylase-treated (F1-met) fragments were digested with methylation-sensitive BstUI and analyzed on polyacrylamide gels to verify efficiency of in vitro methylation. ( C ) Control unmethylated (F1) or SssI-methylated (F1-met) fragments were analyzed by gel-shift assay (EMSA). F, free probe; B, CTCF-bound probe. ( D ) Representation of the hTERT sequence cloned into the pTERT-297/ex2/FRT. Arrows represent the localization of the primers used for hTERT methylation analysis of stable transfectant. ( E ) The methylation status of the stable transfectants was verified by MS-SSCA. Unmethylated and fully methylated controls were obtained from plasmids used for stable <t>transfection.</t> UT and MT represent, respectively, the unmethylated and methylated plasmids stably transfected into HeLa cells, and after 30 population doublings. ( F ) Binding of CTCF to the first exon of hTERT in stably transfected cell line was analyzed by ChIP assay using anti-CTCF antibody.
    Transient Transfection Hcfs, supplied by ScienCell, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection hcfs/product/ScienCell
    Average 90 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    transient transfection hcfs - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    99
    Qiagen transient transfection
    Inhibition of encystation by AcAtg3-siRNA. Cells transfected with Superfect <t>Transfection</t> reagent, nonspecific negative siRNA, or AcAtg3-siRNA were incubated in encystment media for 3 days and the number of mature cysts scored. The formation of mature cysts was almost inhibited in AcAtg3-siRNA transfected cells.
    Transient Transfection, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 3504 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection/product/Qiagen
    Average 99 stars, based on 3504 article reviews
    Price from $9.99 to $1999.99
    transient transfection - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    80
    Stratagene transient transfections psg5
    Inhibition of encystation by AcAtg3-siRNA. Cells transfected with Superfect <t>Transfection</t> reagent, nonspecific negative siRNA, or AcAtg3-siRNA were incubated in encystment media for 3 days and the number of mature cysts scored. The formation of mature cysts was almost inhibited in AcAtg3-siRNA transfected cells.
    Transient Transfections Psg5, supplied by Stratagene, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfections psg5/product/Stratagene
    Average 80 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    transient transfections psg5 - by Bioz Stars, 2020-08
    80/100 stars
      Buy from Supplier

    86
    Fisher Scientific transient transfections glass coverslips
    Inhibition of encystation by AcAtg3-siRNA. Cells transfected with Superfect <t>Transfection</t> reagent, nonspecific negative siRNA, or AcAtg3-siRNA were incubated in encystment media for 3 days and the number of mature cysts scored. The formation of mature cysts was almost inhibited in AcAtg3-siRNA transfected cells.
    Transient Transfections Glass Coverslips, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfections glass coverslips/product/Fisher Scientific
    Average 86 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    transient transfections glass coverslips - by Bioz Stars, 2020-08
    86/100 stars
      Buy from Supplier

    91
    Polypus Transfection transient transfection utilized jetprime
    Inhibition of encystation by AcAtg3-siRNA. Cells transfected with Superfect <t>Transfection</t> reagent, nonspecific negative siRNA, or AcAtg3-siRNA were incubated in encystment media for 3 days and the number of mature cysts scored. The formation of mature cysts was almost inhibited in AcAtg3-siRNA transfected cells.
    Transient Transfection Utilized Jetprime, supplied by Polypus Transfection, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection utilized jetprime/product/Polypus Transfection
    Average 91 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    transient transfection utilized jetprime - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    92
    GE Healthcare transient transfection dharmafect 4 transfection reagent
    Inhibition of encystation by AcAtg3-siRNA. Cells transfected with Superfect <t>Transfection</t> reagent, nonspecific negative siRNA, or AcAtg3-siRNA were incubated in encystment media for 3 days and the number of mature cysts scored. The formation of mature cysts was almost inhibited in AcAtg3-siRNA transfected cells.
    Transient Transfection Dharmafect 4 Transfection Reagent, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transient transfection dharmafect 4 transfection reagent/product/GE Healthcare
    Average 92 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    transient transfection dharmafect 4 transfection reagent - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    Image Search Results


    Site-directed mutagenesis for luciferase activity assay and effect of miR-7 silencing over MAFG expression. (A) Chromosomal localization of miR-7 predicted binding sites at 3'UTR of MAFG. Regions 2 and 8 were identified by six or more bioinformatical algorithms. Sanger sequencing showed that the seed sequence of miR-7 was fully mutated at regions 2 and 8 of the 3' UTR of MAFG. (B) Co-transfection of mimic miR-7 (miR-7) or mimic control (miR-NC) with the 3' UTR of MAFG WT, mutated on region 2 (MAFG 2*) and mutated on region 8 (MAFG 8*). Experiments were performed at 15nM and data was analyzed after 24h of co-transfection. (Upper panel) Relative luciferase activity. The figures represent the mean ± SD of at least 3 independent experiments after data normalization with Renilla and the data from the negative control 3'-UTR; p

    Journal: Theranostics

    Article Title: DNA Methylation of miR-7 is a Mechanism Involved in Platinum Response through MAFG Overexpression in Cancer Cells

    doi: 10.7150/thno.20112

    Figure Lengend Snippet: Site-directed mutagenesis for luciferase activity assay and effect of miR-7 silencing over MAFG expression. (A) Chromosomal localization of miR-7 predicted binding sites at 3'UTR of MAFG. Regions 2 and 8 were identified by six or more bioinformatical algorithms. Sanger sequencing showed that the seed sequence of miR-7 was fully mutated at regions 2 and 8 of the 3' UTR of MAFG. (B) Co-transfection of mimic miR-7 (miR-7) or mimic control (miR-NC) with the 3' UTR of MAFG WT, mutated on region 2 (MAFG 2*) and mutated on region 8 (MAFG 8*). Experiments were performed at 15nM and data was analyzed after 24h of co-transfection. (Upper panel) Relative luciferase activity. The figures represent the mean ± SD of at least 3 independent experiments after data normalization with Renilla and the data from the negative control 3'-UTR; p

    Article Snippet: cDNA plasmids transfection A Myc-DDK-tagged ORF clone of MAFG , ELK-1 or ABCA1 and the negative control pCMV6 were used for in transient transfection (OriGene, USA).

    Techniques: Mutagenesis, Luciferase, Activity Assay, Expressing, Binding Assay, Sequencing, Cotransfection, Negative Control

    Mapping of the RNA sequence(s) in HBV pregenomic RNA responsible for HBV RNA decay. (A) Schematic diagram of the truncation and deletion mutants of HBV pregenomic RNA. The numbers indicate the HBV DNA sequence with 1 at the unique EcoRI site in the HBV genome. (B) Huh7 cells were transfected with 0.1 μg of luciferase fusion constructs containing the truncation mutants of HBV DNA and 0.2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h, and the luciferase activity was then assessed. For each transfection, 0.01 μg of pRL-tk was included as an internal control of transfection efficiency. Results represent the means of data from three independent experiments performed in duplicate. *, P

    Journal: Journal of Virology

    Article Title: Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs ▿

    doi: 10.1128/JVI.00236-10

    Figure Lengend Snippet: Mapping of the RNA sequence(s) in HBV pregenomic RNA responsible for HBV RNA decay. (A) Schematic diagram of the truncation and deletion mutants of HBV pregenomic RNA. The numbers indicate the HBV DNA sequence with 1 at the unique EcoRI site in the HBV genome. (B) Huh7 cells were transfected with 0.1 μg of luciferase fusion constructs containing the truncation mutants of HBV DNA and 0.2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h, and the luciferase activity was then assessed. For each transfection, 0.01 μg of pRL-tk was included as an internal control of transfection efficiency. Results represent the means of data from three independent experiments performed in duplicate. *, P

    Article Snippet: Transient transfection was performed by using FuGENE 6 reagent (Roche) according to the manufacturer's instructions.

    Techniques: Sequencing, Transfection, Luciferase, Construct, Activity Assay

    The decay of HBV pre-S2/S RNA in the nucleus is associated with the deficiency in PRE-dependent nuclear transport. (A) Huh7 cells were transfected with 0.2 μg of pRSV-CAT or pRSV138PRE-CAT together with 0.4 μg of pCMV/Myc or pCMV/Myc-MyD88. At 48 h posttransfection, the cells were harvested, and CAT activity was measured. For each transfection, 0.1 μg of β-Gal was included as an internal control of transfection efficiency. Results represent the means of data from three independent experiments performed in duplicate. *, P

    Journal: Journal of Virology

    Article Title: Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs ▿

    doi: 10.1128/JVI.00236-10

    Figure Lengend Snippet: The decay of HBV pre-S2/S RNA in the nucleus is associated with the deficiency in PRE-dependent nuclear transport. (A) Huh7 cells were transfected with 0.2 μg of pRSV-CAT or pRSV138PRE-CAT together with 0.4 μg of pCMV/Myc or pCMV/Myc-MyD88. At 48 h posttransfection, the cells were harvested, and CAT activity was measured. For each transfection, 0.1 μg of β-Gal was included as an internal control of transfection efficiency. Results represent the means of data from three independent experiments performed in duplicate. *, P

    Article Snippet: Transient transfection was performed by using FuGENE 6 reagent (Roche) according to the manufacturer's instructions.

    Techniques: Transfection, Activity Assay

    MyD88 inhibits HBV replication in HepG2.2.15 cells and in a mouse model. (A) HepG2.2.15 cells were mock infected or infected with Ad-EGFP or Ad-MyD88 or treated with 5,000 IU/ml IFN-α. The levels of viral RNA and core particle-associated DNA were determined by Northern (top) and Southern (middle) blot analyses, respectively, using a 32 P-radiolabeled HBV DNA probe. After the detection of HBV RNA, the blots were stripped and rehybridized with a 32 P-radiolabeled GAPDH DNA probe to control for gel loading. The positions of HBV pregenomic RNA (3.5 kb), pre-S1/S RNA (2.4 kb), and pre-S2/S RNA (2.1 kb) and the positions of relaxed circular (RC), single-stranded (SS) DNAs are indicated. MyD88 expression was confirmed by Western blot analysis using an anti-MyD88 antibody. The levels of the β-actin protein were determined to normalize for protein loading (bottom). (B) BALB/c mice were hydrodynamically coinjected with pHBV1.3 and pCMV/Myc or pCMV/Myc-MyD88. At 4 days postinjection, total liver DNA was extracted and subjected to digestion with HindIII restriction endonuclease. Input pHBV1.3 and viral core particle-associated DNA were examined by Southern blot analysis (top). The intensity of input pHBV1.3 in each lane was used as an internal control indicating equivalent transfection efficiency. Total liver RNA was analyzed by Northern blotting, and GAPDH was analyzed as a loading control (middle). MyD88 expression was confirmed by Western blot analysis using an anti-Myc antibody (bottom). Eight comparable pairs in which each part showed similar amounts of input DNA were obtained from two independent injections, and two representative pairs are shown. (C) Huh7 (top) and HepG2 (bottom) cells were cotransfected with the indicated amounts of pCIdA-HBV and pCMV/Myc or pCMV/Myc-MyD88. Levels of HBV RNAs were determined by Northern blot analysis. GAPDH was analyzed as a loading control.

    Journal: Journal of Virology

    Article Title: Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs ▿

    doi: 10.1128/JVI.00236-10

    Figure Lengend Snippet: MyD88 inhibits HBV replication in HepG2.2.15 cells and in a mouse model. (A) HepG2.2.15 cells were mock infected or infected with Ad-EGFP or Ad-MyD88 or treated with 5,000 IU/ml IFN-α. The levels of viral RNA and core particle-associated DNA were determined by Northern (top) and Southern (middle) blot analyses, respectively, using a 32 P-radiolabeled HBV DNA probe. After the detection of HBV RNA, the blots were stripped and rehybridized with a 32 P-radiolabeled GAPDH DNA probe to control for gel loading. The positions of HBV pregenomic RNA (3.5 kb), pre-S1/S RNA (2.4 kb), and pre-S2/S RNA (2.1 kb) and the positions of relaxed circular (RC), single-stranded (SS) DNAs are indicated. MyD88 expression was confirmed by Western blot analysis using an anti-MyD88 antibody. The levels of the β-actin protein were determined to normalize for protein loading (bottom). (B) BALB/c mice were hydrodynamically coinjected with pHBV1.3 and pCMV/Myc or pCMV/Myc-MyD88. At 4 days postinjection, total liver DNA was extracted and subjected to digestion with HindIII restriction endonuclease. Input pHBV1.3 and viral core particle-associated DNA were examined by Southern blot analysis (top). The intensity of input pHBV1.3 in each lane was used as an internal control indicating equivalent transfection efficiency. Total liver RNA was analyzed by Northern blotting, and GAPDH was analyzed as a loading control (middle). MyD88 expression was confirmed by Western blot analysis using an anti-Myc antibody (bottom). Eight comparable pairs in which each part showed similar amounts of input DNA were obtained from two independent injections, and two representative pairs are shown. (C) Huh7 (top) and HepG2 (bottom) cells were cotransfected with the indicated amounts of pCIdA-HBV and pCMV/Myc or pCMV/Myc-MyD88. Levels of HBV RNAs were determined by Northern blot analysis. GAPDH was analyzed as a loading control.

    Article Snippet: Transient transfection was performed by using FuGENE 6 reagent (Roche) according to the manufacturer's instructions.

    Techniques: Infection, Northern Blot, Expressing, Western Blot, Mouse Assay, Southern Blot, Transfection

    MyD88 downregulates HBV RNAs by a posttranscriptional mechanism. (A) Huh7 cells were cotransfected with 0.1 μg of each of four reporter plasmids for HBV promoters and enhancers (ENII/Cp, Sp1, Sp2, and ENI/Xp, respectively) and pCMV/Myc or pCMV/Myc-MyD88. The cells were harvested at 48 h posttransfection, and luciferase activity in the lysates was assessed. For each transfection, 0.01 μg of pRL-tk was included as an internal control of transfection efficiency. Results represent the means of data from three independent experiments performed in duplicate. (B). Huh7 cells were cotransfected with the indicated amounts of pCMV-HBV and pCMV/Myc or pCMV/Myc-MyD88. Levels of the pregenomic RNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control. (C) Huh7 cells were cotransfected with the indicated amounts of pCDNA3.1-Luc and pCMV/Myc or pCMV/Myc-MyD88. Levels of luciferase mRNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control. (D) Vero cells were transfected as described above (B). Levels of pregenomic RNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control.

    Journal: Journal of Virology

    Article Title: Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs ▿

    doi: 10.1128/JVI.00236-10

    Figure Lengend Snippet: MyD88 downregulates HBV RNAs by a posttranscriptional mechanism. (A) Huh7 cells were cotransfected with 0.1 μg of each of four reporter plasmids for HBV promoters and enhancers (ENII/Cp, Sp1, Sp2, and ENI/Xp, respectively) and pCMV/Myc or pCMV/Myc-MyD88. The cells were harvested at 48 h posttransfection, and luciferase activity in the lysates was assessed. For each transfection, 0.01 μg of pRL-tk was included as an internal control of transfection efficiency. Results represent the means of data from three independent experiments performed in duplicate. (B). Huh7 cells were cotransfected with the indicated amounts of pCMV-HBV and pCMV/Myc or pCMV/Myc-MyD88. Levels of the pregenomic RNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control. (C) Huh7 cells were cotransfected with the indicated amounts of pCDNA3.1-Luc and pCMV/Myc or pCMV/Myc-MyD88. Levels of luciferase mRNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control. (D) Vero cells were transfected as described above (B). Levels of pregenomic RNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control.

    Article Snippet: Transient transfection was performed by using FuGENE 6 reagent (Roche) according to the manufacturer's instructions.

    Techniques: Luciferase, Activity Assay, Transfection, Northern Blot

    MyD88 accelerates the decay of HBV pregenomic RNA in the cytoplasm. (A) Huh7 cells were cotransfected with pTet-HBV and pUHD-TA together with pCMV/Myc or pCMV/Myc-MyD88. At 39 h posttransfection, the cells were harvested directly upon the addition of doxycycline (Dox) or at 3, 6, or 9 h thereafter. The levels of pregenomic RNA were determined by Northern blot analysis. To normalize RNA loading, the same blots were hybridized with a GAPDH probe. (B) The half-life of the pregenomic RNA was determined by Northern blot analysis, and the bands were quantified by phosphorimaging. Two independent experiments performed in duplicate were analyzed, and the levels of pregenomic RNA were normalized against RNA loading. (C) Huh7 cells were transfected as described above (A). Cytoplasmic (left) and nuclear (right) RNAs were prepared and analyzed by Northern blotting. (D and E) The half-lives of pregenomic RNA in the cytoplasm (D) and nucleus (E) were determined by Northern blot analysis, and the bands were quantified by phosphorimaging. Two independent experiments performed in duplicate were analyzed and quantified by phosphorimaging, and the levels of pregenomic RNA were normalized against RNA loading. (F) Huh7 cells were transfected with 40 nM siRNA targeting EGFP, DCP2, or EXOSC5. At 24 h posttransfection, the cells were transfected with 20 nM the same siRNA together with pHBV1.3 and pCMV/Myc-MyD88. At 48 h after the second transfection, the cells were harvested, and viral pregenomic RNA was analyzed by Northern blot analysis (top). The expression of target proteins was evaluated by Western blot analysis using anti-DCP2 and anti-EXOSC5 antibodies (bottom).

    Journal: Journal of Virology

    Article Title: Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs ▿

    doi: 10.1128/JVI.00236-10

    Figure Lengend Snippet: MyD88 accelerates the decay of HBV pregenomic RNA in the cytoplasm. (A) Huh7 cells were cotransfected with pTet-HBV and pUHD-TA together with pCMV/Myc or pCMV/Myc-MyD88. At 39 h posttransfection, the cells were harvested directly upon the addition of doxycycline (Dox) or at 3, 6, or 9 h thereafter. The levels of pregenomic RNA were determined by Northern blot analysis. To normalize RNA loading, the same blots were hybridized with a GAPDH probe. (B) The half-life of the pregenomic RNA was determined by Northern blot analysis, and the bands were quantified by phosphorimaging. Two independent experiments performed in duplicate were analyzed, and the levels of pregenomic RNA were normalized against RNA loading. (C) Huh7 cells were transfected as described above (A). Cytoplasmic (left) and nuclear (right) RNAs were prepared and analyzed by Northern blotting. (D and E) The half-lives of pregenomic RNA in the cytoplasm (D) and nucleus (E) were determined by Northern blot analysis, and the bands were quantified by phosphorimaging. Two independent experiments performed in duplicate were analyzed and quantified by phosphorimaging, and the levels of pregenomic RNA were normalized against RNA loading. (F) Huh7 cells were transfected with 40 nM siRNA targeting EGFP, DCP2, or EXOSC5. At 24 h posttransfection, the cells were transfected with 20 nM the same siRNA together with pHBV1.3 and pCMV/Myc-MyD88. At 48 h after the second transfection, the cells were harvested, and viral pregenomic RNA was analyzed by Northern blot analysis (top). The expression of target proteins was evaluated by Western blot analysis using anti-DCP2 and anti-EXOSC5 antibodies (bottom).

    Article Snippet: Transient transfection was performed by using FuGENE 6 reagent (Roche) according to the manufacturer's instructions.

    Techniques: Northern Blot, Transfection, Expressing, Western Blot

    Promoter activities of ALOX15 variants in NIH3T3 cells and MCF-7 cells. Vimentin binds to ALOX15 promoter ( A ) ALOX15 promoter sequence. Primers used for this study and alternative alleles of rs2255888 are shown in bold. (G/A) in bold denotes SNP rs2255888 position. ( B ) Qualitative measurement of human ALOX15 (upper panel) and GAPDH (lower panel) gene expression by RT-PCR in NIH3T3 cells. ( C ) Luciferase activities of P1 and P2 transfected NIH3T3 cells was measured after 24 h. *denotes p = 2×10 −5 vs. P2. The results are the average of two independent transfections performed in triplicate ±S.E. ( D ) Human ALOX15 (upper panel) and GAPDH (lower panel) gene expression by RT-PCR in MCF-7 cells in presence of vimentin. ( E ) Luciferase activities of P1 and P2 transfected MCF-7 cells were measured after 24 h. The experiments were done in triplicates. **denotes p

    Journal: PLoS ONE

    Article Title: Characterization of a Human 12/15-Lipoxygenase Promoter Variant Associated with Atherosclerosis Identifies Vimentin as a Promoter Binding Protein

    doi: 10.1371/journal.pone.0042417

    Figure Lengend Snippet: Promoter activities of ALOX15 variants in NIH3T3 cells and MCF-7 cells. Vimentin binds to ALOX15 promoter ( A ) ALOX15 promoter sequence. Primers used for this study and alternative alleles of rs2255888 are shown in bold. (G/A) in bold denotes SNP rs2255888 position. ( B ) Qualitative measurement of human ALOX15 (upper panel) and GAPDH (lower panel) gene expression by RT-PCR in NIH3T3 cells. ( C ) Luciferase activities of P1 and P2 transfected NIH3T3 cells was measured after 24 h. *denotes p = 2×10 −5 vs. P2. The results are the average of two independent transfections performed in triplicate ±S.E. ( D ) Human ALOX15 (upper panel) and GAPDH (lower panel) gene expression by RT-PCR in MCF-7 cells in presence of vimentin. ( E ) Luciferase activities of P1 and P2 transfected MCF-7 cells were measured after 24 h. The experiments were done in triplicates. **denotes p

    Article Snippet: Transient Transfection Transient transfection of NIH3T3 cells with cloned luciferase constructs (P1 and P2) were carried out with Fugene 6 (Roche Applied Science, Indianapolis, IN) according to manufacturer’s protocol.

    Techniques: Sequencing, Expressing, Reverse Transcription Polymerase Chain Reaction, Luciferase, Transfection

    Relative change in TAG- or CE-specific intracellular lipid storage upon expression of various Plin proteins. AML12 cells were transiently transfected with the indicated GFP–Plin-expressing constructs and cultured overnight in the presence of oleic acid and cholesterol. Transfection efficiencies were confirmed by visualizing GFP fluorescence. Untransfected cells, cultured with/without exogenous oleic acid and cholesterol, were grown in parallel. LSDs were isolated by centrifugation and lipids were extracted, separated by TLC in parallel with lipid markers, and TAG and CE detected after staining with iodine vapor. Relative TAG/CE ratios were quantified in cells transfected with each specific Plin-expressing construct and analyzed in parallel with identically grown untransfected cells for normalization and TLC background correction. The TAG/CE ratio for untransfected lipid-loaded cells (controls) was set to 0.5. The relative TAG/CE-ratio of Plin1a- and Plin1c-expressing cells were always analyzed in parallel and normalized to those of control cells, and then secondarily compared to results determined for its Plin-expressing counterpart. Values > 0.5 indicate a proportional increase in TAG lipid bias, whereas values

    Journal: Journal of Cell Science

    Article Title: Perilipin family members preferentially sequester to either triacylglycerol-specific or cholesteryl-ester-specific intracellular lipid storage droplets

    doi: 10.1242/jcs.104943

    Figure Lengend Snippet: Relative change in TAG- or CE-specific intracellular lipid storage upon expression of various Plin proteins. AML12 cells were transiently transfected with the indicated GFP–Plin-expressing constructs and cultured overnight in the presence of oleic acid and cholesterol. Transfection efficiencies were confirmed by visualizing GFP fluorescence. Untransfected cells, cultured with/without exogenous oleic acid and cholesterol, were grown in parallel. LSDs were isolated by centrifugation and lipids were extracted, separated by TLC in parallel with lipid markers, and TAG and CE detected after staining with iodine vapor. Relative TAG/CE ratios were quantified in cells transfected with each specific Plin-expressing construct and analyzed in parallel with identically grown untransfected cells for normalization and TLC background correction. The TAG/CE ratio for untransfected lipid-loaded cells (controls) was set to 0.5. The relative TAG/CE-ratio of Plin1a- and Plin1c-expressing cells were always analyzed in parallel and normalized to those of control cells, and then secondarily compared to results determined for its Plin-expressing counterpart. Values > 0.5 indicate a proportional increase in TAG lipid bias, whereas values

    Article Snippet: Transient transfection was carried out following the manufacturer's instructions (Invitrogen) using Lipofectamine LTX reagent.

    Techniques: Expressing, Transfection, Construct, Cell Culture, Fluorescence, Isolation, Centrifugation, Thin Layer Chromatography, Staining

    Choline kinase regulates Akt activity . A, MDA-MB 468 cells were transfected with 50 nM of the indicated pool siRNA. 3 days posttransfection, 50 μM LY294002 was added to scr transfected cells for 30 mins. Whole cell lysate were subjected to western blotting with the indicated antibodies. B, siRNA transfection and western blot were performed as (A). Percentage of pAkt(ser473) signal were calculated by quantifying the pAkt(ser473) normalized to total Akt on the immunoblot and compared to the scr control (set as 100%). C, MDA-MB 231 cells were transfected with 50 nM of indicated pool siRNA for 2 days. Cells were serum-starved overnight and stimulated with IGF for 15 mins. Whole cell lysates were harvested and western blot performed with the indicated antibodies. The values below the blot indicate the ratio of normalized pAkt(ser473) signals quantified using Image J to that of scr control (set as 1). D, MCF7 were transfected with 1 μg pcDNA vector, ChoK A or B plasmids for 24 h using Lipofectamine 2000 (Invitrogen). Cells were harvested and ChoK activity determined as described in the methods. E, MCF7 was transfected as in (D) and western blot with indicated antibodies.

    Journal: Molecular Cancer

    Article Title: Regulation of Akt(ser473) phosphorylation by Choline kinase in breast carcinoma cells

    doi: 10.1186/1476-4598-8-131

    Figure Lengend Snippet: Choline kinase regulates Akt activity . A, MDA-MB 468 cells were transfected with 50 nM of the indicated pool siRNA. 3 days posttransfection, 50 μM LY294002 was added to scr transfected cells for 30 mins. Whole cell lysate were subjected to western blotting with the indicated antibodies. B, siRNA transfection and western blot were performed as (A). Percentage of pAkt(ser473) signal were calculated by quantifying the pAkt(ser473) normalized to total Akt on the immunoblot and compared to the scr control (set as 100%). C, MDA-MB 231 cells were transfected with 50 nM of indicated pool siRNA for 2 days. Cells were serum-starved overnight and stimulated with IGF for 15 mins. Whole cell lysates were harvested and western blot performed with the indicated antibodies. The values below the blot indicate the ratio of normalized pAkt(ser473) signals quantified using Image J to that of scr control (set as 1). D, MCF7 were transfected with 1 μg pcDNA vector, ChoK A or B plasmids for 24 h using Lipofectamine 2000 (Invitrogen). Cells were harvested and ChoK activity determined as described in the methods. E, MCF7 was transfected as in (D) and western blot with indicated antibodies.

    Article Snippet: Transfection Transient transfections using Lipofectamine™ 2000 (Invitrogen) were performed following the manufacturer's instructions.

    Techniques: Activity Assay, Multiple Displacement Amplification, Transfection, Western Blot, Plasmid Preparation

    ChoKα regulation on Akt activity is PI3K independent . MDA-MB 231 and A549 cells were transfected with 50 nM of pool siRNA targeted to scr control or ChoK A. 2 days post transfection, cells were transfected with 0.5 μg PH-GFP construct for 24 h followed by 8 h serum starvation and 15 mins of IGF stimulation. 50 μM LY294002 was added to scr transfected cells for 30 mins prior to IGF stimulation. Cells were stained with Hoechst, fixed with paraformaldehyde and mounted for fluorescence microscopy.

    Journal: Molecular Cancer

    Article Title: Regulation of Akt(ser473) phosphorylation by Choline kinase in breast carcinoma cells

    doi: 10.1186/1476-4598-8-131

    Figure Lengend Snippet: ChoKα regulation on Akt activity is PI3K independent . MDA-MB 231 and A549 cells were transfected with 50 nM of pool siRNA targeted to scr control or ChoK A. 2 days post transfection, cells were transfected with 0.5 μg PH-GFP construct for 24 h followed by 8 h serum starvation and 15 mins of IGF stimulation. 50 μM LY294002 was added to scr transfected cells for 30 mins prior to IGF stimulation. Cells were stained with Hoechst, fixed with paraformaldehyde and mounted for fluorescence microscopy.

    Article Snippet: Transfection Transient transfections using Lipofectamine™ 2000 (Invitrogen) were performed following the manufacturer's instructions.

    Techniques: Activity Assay, Multiple Displacement Amplification, Transfection, Construct, Staining, Fluorescence, Microscopy

    Outline of pharmacological manipulation of CK1 functions and its effects on p53 signalling pathway, cell viability, and CK1α post-translational modifications. ( A ) H1299 cells were transfected with STReP-tagged CK1α sp1 or 2, FLAG-tagged peptide 35 and His-Ubiquitin or His-Nedd8, using Attractene. Eighteen hours after transfection cells were treated with 50 µM MG132 for 4 hours. Whole cell lysates were analysed for transfected CK1α levels, and His-ubiquitin/NEDD8 conjugates were purified by metal affinity chromatography and analyzed by 4%–12% NuPAGE/immunoblots with an anti-CK1α antibody. Changes highlighted were representative of two independent experiments. ( B ) Three approaches for manipulation of signal transduction pathways. Genetic depletion of CK1α using siRNA or the CK1 kinase attenuation using the ATP-competitive inhibitor D4476 can alter the p53 response and cell viability, as indicated. The bioactive peptide from the high affinity MDM2-CK1α interface (peptide 35) highlights a novel peptide lead for disrupting signalling in cancers. All three approaches gave rise to overlapping but distinct effects on signalling. Specific siRNA against CK1α led to p53 induction and growth arrest accompanied with slight sub-G1 increase, whereas non-specific CK1 drugs such as D4476 mediated p53 induction and significant apoptosis. Low levels of the bioactive peptide 35 did not lead to p53 protein induction but to a growth arrest in G0-G1 and reduced cell viability. These data suggest that peptide 35 may function in a pharmacologically novel manner compared to the ATP-active site inhibitor or CK1α siRNA and highlights the general utility of targeting protein-protein interactions as approaches for developing therapeutic strategies that target signalling mechanisms in cancer.

    Journal: PLoS ONE

    Article Title: Exploiting the MDM2-CK1? Protein-Protein Interface to Develop Novel Biologics That Induce UBL-Kinase-Modification and Inhibit Cell Growth

    doi: 10.1371/journal.pone.0043391

    Figure Lengend Snippet: Outline of pharmacological manipulation of CK1 functions and its effects on p53 signalling pathway, cell viability, and CK1α post-translational modifications. ( A ) H1299 cells were transfected with STReP-tagged CK1α sp1 or 2, FLAG-tagged peptide 35 and His-Ubiquitin or His-Nedd8, using Attractene. Eighteen hours after transfection cells were treated with 50 µM MG132 for 4 hours. Whole cell lysates were analysed for transfected CK1α levels, and His-ubiquitin/NEDD8 conjugates were purified by metal affinity chromatography and analyzed by 4%–12% NuPAGE/immunoblots with an anti-CK1α antibody. Changes highlighted were representative of two independent experiments. ( B ) Three approaches for manipulation of signal transduction pathways. Genetic depletion of CK1α using siRNA or the CK1 kinase attenuation using the ATP-competitive inhibitor D4476 can alter the p53 response and cell viability, as indicated. The bioactive peptide from the high affinity MDM2-CK1α interface (peptide 35) highlights a novel peptide lead for disrupting signalling in cancers. All three approaches gave rise to overlapping but distinct effects on signalling. Specific siRNA against CK1α led to p53 induction and growth arrest accompanied with slight sub-G1 increase, whereas non-specific CK1 drugs such as D4476 mediated p53 induction and significant apoptosis. Low levels of the bioactive peptide 35 did not lead to p53 protein induction but to a growth arrest in G0-G1 and reduced cell viability. These data suggest that peptide 35 may function in a pharmacologically novel manner compared to the ATP-active site inhibitor or CK1α siRNA and highlights the general utility of targeting protein-protein interactions as approaches for developing therapeutic strategies that target signalling mechanisms in cancer.

    Article Snippet: Transient Transfection of Small Interfering RNA (siRNA) siRNA to CK1α gene was obtained from Dharmacon (siGENOME SMARTpool against Human CSNK1A1 (NM_001892)). siCONTROL non-targeting siRNA Pool#2 was used as a control (siRNA control).

    Techniques: Transfection, Purification, Affinity Chromatography, Western Blot, Transduction

    CK1α peptide derived from its dominant MDM2 binding site triggers G0-G1 arrest and cell death in a p53-independent manner. CK1α peptide 35 was transfected into HCT116 cells using Nucleofectin reagent and Nucleofector device II. A DMSO control was included. ( A ) Protein levels were assessed 18 hours after peptide transfection by Western blotting. ( B ) Images of cells were captured 16 hours after transfection. ( C ) The number of non-viable cells was counted after treatment using Trypan Blue. The percentage of non-viable cells is expressed relative to each respective total cell count. ( D ) After treatment, the cells were harvested then fixed in ethanol followed by staining with propidium iodide. DNA content was determined by FACS and analyzed with FlowJo7 software.

    Journal: PLoS ONE

    Article Title: Exploiting the MDM2-CK1? Protein-Protein Interface to Develop Novel Biologics That Induce UBL-Kinase-Modification and Inhibit Cell Growth

    doi: 10.1371/journal.pone.0043391

    Figure Lengend Snippet: CK1α peptide derived from its dominant MDM2 binding site triggers G0-G1 arrest and cell death in a p53-independent manner. CK1α peptide 35 was transfected into HCT116 cells using Nucleofectin reagent and Nucleofector device II. A DMSO control was included. ( A ) Protein levels were assessed 18 hours after peptide transfection by Western blotting. ( B ) Images of cells were captured 16 hours after transfection. ( C ) The number of non-viable cells was counted after treatment using Trypan Blue. The percentage of non-viable cells is expressed relative to each respective total cell count. ( D ) After treatment, the cells were harvested then fixed in ethanol followed by staining with propidium iodide. DNA content was determined by FACS and analyzed with FlowJo7 software.

    Article Snippet: Transient Transfection of Small Interfering RNA (siRNA) siRNA to CK1α gene was obtained from Dharmacon (siGENOME SMARTpool against Human CSNK1A1 (NM_001892)). siCONTROL non-targeting siRNA Pool#2 was used as a control (siRNA control).

    Techniques: Derivative Assay, Binding Assay, Transfection, Western Blot, Cell Counting, Staining, FACS, Software

    The CK1α peptide derived from its dominant MDM2 binding site triggers G0-G1 arrest and cell death in A375 cells. CK1α peptide 35 was transfected into A375 cells using Nucleofectin reagent and Nucleofector device II. A DMSO control was included. ( A ) Protein levels were assessed 18 hours after peptide transfection by Western blotting. ( B ) Images of cells were captured 16 hours after transfection. ( C ) The number of non-viable cells was counted after treatment using Trypan Blue. Total cells are shown in percent relative to the DMSO control and percent of non-viable cells are expressed relative to each respective total cell count. ( D ) After treatment, the cells were harvested then fixed in ethanol followed by staining with propidium iodide. DNA content was determined by FACS and analyzed with FlowJo7 software.

    Journal: PLoS ONE

    Article Title: Exploiting the MDM2-CK1? Protein-Protein Interface to Develop Novel Biologics That Induce UBL-Kinase-Modification and Inhibit Cell Growth

    doi: 10.1371/journal.pone.0043391

    Figure Lengend Snippet: The CK1α peptide derived from its dominant MDM2 binding site triggers G0-G1 arrest and cell death in A375 cells. CK1α peptide 35 was transfected into A375 cells using Nucleofectin reagent and Nucleofector device II. A DMSO control was included. ( A ) Protein levels were assessed 18 hours after peptide transfection by Western blotting. ( B ) Images of cells were captured 16 hours after transfection. ( C ) The number of non-viable cells was counted after treatment using Trypan Blue. Total cells are shown in percent relative to the DMSO control and percent of non-viable cells are expressed relative to each respective total cell count. ( D ) After treatment, the cells were harvested then fixed in ethanol followed by staining with propidium iodide. DNA content was determined by FACS and analyzed with FlowJo7 software.

    Article Snippet: Transient Transfection of Small Interfering RNA (siRNA) siRNA to CK1α gene was obtained from Dharmacon (siGENOME SMARTpool against Human CSNK1A1 (NM_001892)). siCONTROL non-targeting siRNA Pool#2 was used as a control (siRNA control).

    Techniques: Derivative Assay, Binding Assay, Transfection, Western Blot, Cell Counting, Staining, FACS, Software

    Stimulation-induced processing and degradation of p105 is not affected by mutating lysine residues 18 (683) to 30 (967) in the C-terminal domain of the molecule. (A) COS-7 cells were transiently transfected with either a cDNA coding for p105-WT (lanes 2 to 5) or p105-K18-30R (lanes 6 to 9) as indicated. Control cells (lane 1) were transfected with an empty vector. Where indicated, a cDNA coding for constitutively active IKKβ was cotransfected. Twenty-four hours after transfection cells were harvested, and nuclear and cytosolic fractions were isolated as described in Materials and Methods. Aliquots of cytosolic and nuclear extracts representing an equal number of cells were resolved via SDS-10% PAGE and were blotted onto nitrocellulose paper, and proteins were visualized by using anti-p50 antibody and ECL as described in Materials and Methods. C, cytosolic fraction; N, nuclear fraction. (B) COS-7 cells were transiently transfected with a cDNA coding for either p105-WT (lanes 1 to 4), p105-K18-30R (lanes 5 to 8), or p105-Δ917-932 (lanes 9 to 12). Where indicated, cDNA coding for a constitutively active IKKβ was cotransfected. Twenty-four hours after transfection cells were pulse labeled with [ 35 S]methionine (0; pulse). Following removal and dilution of the labeled amino acid and further incubation for 2 h (2; chase), the labeled proteins were immunoprecipitated by using anti-p50 antibody, resolved by SDS-10% PAGE, and visualized by phosphorimaging as described in Materials and Methods.

    Journal: Molecular and Cellular Biology

    Article Title: Dual Effects of I?B Kinase ?-Mediated Phosphorylation on p105 Fate: SCFβ-TrCP-Dependent Degradation and SCFβ-TrCP-Independent Processing

    doi: 10.1128/MCB.24.1.475-486.2004

    Figure Lengend Snippet: Stimulation-induced processing and degradation of p105 is not affected by mutating lysine residues 18 (683) to 30 (967) in the C-terminal domain of the molecule. (A) COS-7 cells were transiently transfected with either a cDNA coding for p105-WT (lanes 2 to 5) or p105-K18-30R (lanes 6 to 9) as indicated. Control cells (lane 1) were transfected with an empty vector. Where indicated, a cDNA coding for constitutively active IKKβ was cotransfected. Twenty-four hours after transfection cells were harvested, and nuclear and cytosolic fractions were isolated as described in Materials and Methods. Aliquots of cytosolic and nuclear extracts representing an equal number of cells were resolved via SDS-10% PAGE and were blotted onto nitrocellulose paper, and proteins were visualized by using anti-p50 antibody and ECL as described in Materials and Methods. C, cytosolic fraction; N, nuclear fraction. (B) COS-7 cells were transiently transfected with a cDNA coding for either p105-WT (lanes 1 to 4), p105-K18-30R (lanes 5 to 8), or p105-Δ917-932 (lanes 9 to 12). Where indicated, cDNA coding for a constitutively active IKKβ was cotransfected. Twenty-four hours after transfection cells were pulse labeled with [ 35 S]methionine (0; pulse). Following removal and dilution of the labeled amino acid and further incubation for 2 h (2; chase), the labeled proteins were immunoprecipitated by using anti-p50 antibody, resolved by SDS-10% PAGE, and visualized by phosphorimaging as described in Materials and Methods.

    Article Snippet: The human p105-WT, p105-Δ917-933 (Fig. , respectively), and p105-TthIII 1 (p105 to 544) (the TthIII site is shown on the p105-WT scheme in Fig. ) cDNA constructs used for in vitro translation (in pT7β105) and for transient transfection (in pCI-neo; Promega) in cells were described previously ( - ) and served as a base for further manipulations and expression.

    Techniques: Transfection, Plasmid Preparation, Isolation, Polyacrylamide Gel Electrophoresis, Labeling, Incubation, Immunoprecipitation

    Effects of C/EBPδ knockdown on LPS-induced production of cytokines and chemokines from alveolar macrophage cells. MH-S cells were transiently transfected with 40 nmol/L control siRNA or C/EBPδ siRNA. At 12 hours after transfection, the

    Journal: The American Journal of Pathology

    Article Title: CCAAT/Enhancer-Binding Protein ? Is a Critical Mediator of Lipopolysaccharide-Induced Acute Lung Injury

    doi: 10.1016/j.ajpath.2012.10.013

    Figure Lengend Snippet: Effects of C/EBPδ knockdown on LPS-induced production of cytokines and chemokines from alveolar macrophage cells. MH-S cells were transiently transfected with 40 nmol/L control siRNA or C/EBPδ siRNA. At 12 hours after transfection, the

    Article Snippet: We next evaluated the LPS-induced C/EBP activation in transient transfection assays (Promega) using 2XC/EBP-Luc, a promoter-reporter that contains two copies of a C/EBP binding site, and an expression vector for C/EBPδ.

    Techniques: Transfection

    YFP-N300 downregulates MHC class I expression in M553(R240)tpsn cells M553(R240)Tpsn cells were transiently transfected with YFP-N300 or co-transfected with empty vector and pmaxGFP. Twenty-four hours post transfection, MHC class I expression was analyzed by flow cytometry using a PE directly conjugated W6/32 (MHC class I) antibody. Mean fluorescent intensity was determined for W6/32 staining on populations of cells gated on forward and side scatter (R1) and for the negative or positive expression of GFP/YFP (R2, GFP/YFP negative) and R3, (GFP/YFP positive).

    Journal: Human immunology

    Article Title: Identification of an Alternate Splice Form of Tapasin in Human Melanoma

    doi: 10.1016/j.humimm.2010.05.019

    Figure Lengend Snippet: YFP-N300 downregulates MHC class I expression in M553(R240)tpsn cells M553(R240)Tpsn cells were transiently transfected with YFP-N300 or co-transfected with empty vector and pmaxGFP. Twenty-four hours post transfection, MHC class I expression was analyzed by flow cytometry using a PE directly conjugated W6/32 (MHC class I) antibody. Mean fluorescent intensity was determined for W6/32 staining on populations of cells gated on forward and side scatter (R1) and for the negative or positive expression of GFP/YFP (R2, GFP/YFP negative) and R3, (GFP/YFP positive).

    Article Snippet: For transient transfection experiments, 100 ng of the pmaxGFP plasmid which encodes an enhanced GFP protein driven by the CMV promoter (Amaxa, Gaithersburg, MD) and 2 micrograms of the tpsnΔEx3 cDNA in pCDNA3.1(−)PURO or empty pCDNA3.1(−)puro plasmid were transfected into 1 million cells using an Amaxa Nucleoporator II instrument (solution V, program T-030) according to manufacturers recommendations.

    Techniques: Expressing, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Staining

    Transient expression of truncated tapasin molecules does not reduce MHC class I expression in M553(R240)Tpsn cells A . Transient co-transfection of M553(R240)Tpsn cells with pmaxGFP and the indicated tapasin variants followed by W6/32 staining 24 hours post transfection. W6/32 staining on populations of cells gated on forward and side scatter (R1) and for the negative or positive expression of GFP (R2 vs. R3, respectively). B . The transient transfectants from A. were analyzed by western blot for the ratio of wild type (R240)tpsn to tpsnΔEx3 or tpsnΔN50 using TO-3 antibody.

    Journal: Human immunology

    Article Title: Identification of an Alternate Splice Form of Tapasin in Human Melanoma

    doi: 10.1016/j.humimm.2010.05.019

    Figure Lengend Snippet: Transient expression of truncated tapasin molecules does not reduce MHC class I expression in M553(R240)Tpsn cells A . Transient co-transfection of M553(R240)Tpsn cells with pmaxGFP and the indicated tapasin variants followed by W6/32 staining 24 hours post transfection. W6/32 staining on populations of cells gated on forward and side scatter (R1) and for the negative or positive expression of GFP (R2 vs. R3, respectively). B . The transient transfectants from A. were analyzed by western blot for the ratio of wild type (R240)tpsn to tpsnΔEx3 or tpsnΔN50 using TO-3 antibody.

    Article Snippet: For transient transfection experiments, 100 ng of the pmaxGFP plasmid which encodes an enhanced GFP protein driven by the CMV promoter (Amaxa, Gaithersburg, MD) and 2 micrograms of the tpsnΔEx3 cDNA in pCDNA3.1(−)PURO or empty pCDNA3.1(−)puro plasmid were transfected into 1 million cells using an Amaxa Nucleoporator II instrument (solution V, program T-030) according to manufacturers recommendations.

    Techniques: Expressing, Cotransfection, Staining, Transfection, Western Blot

    Dose effect of ENZ fragment on BAR efficiency. ( A ) Schematic representation of the Gal4-tagged (gray–blue square) ORF2 fragments. ENZ490Δ fragment was generated from wild-type ORF2 sequence, while Δ348RTCys fragment was generated using codon optimized ORF2 sequence. ( B ) BAR output using different amounts (0.4–0.05 μg) of the wild-type ENZ expression plasmid with 0.4 μg of the RTCys expression plasmid. Empty vector used as filler to ensure equal DNA amounts in transfections. Error bars denote standard deviation ( n = 3). Statistical significance assessed using Student's t -test for paired samples with denoted by *. Representative flasks are shown above corresponding graph bars.

    Journal: Nucleic Acids Research

    Article Title: Identification of L1 ORF2p sequence important to retrotransposition using Bipartile Alu retrotransposition (BAR)

    doi: 10.1093/nar/gkw277

    Figure Lengend Snippet: Dose effect of ENZ fragment on BAR efficiency. ( A ) Schematic representation of the Gal4-tagged (gray–blue square) ORF2 fragments. ENZ490Δ fragment was generated from wild-type ORF2 sequence, while Δ348RTCys fragment was generated using codon optimized ORF2 sequence. ( B ) BAR output using different amounts (0.4–0.05 μg) of the wild-type ENZ expression plasmid with 0.4 μg of the RTCys expression plasmid. Empty vector used as filler to ensure equal DNA amounts in transfections. Error bars denote standard deviation ( n = 3). Statistical significance assessed using Student's t -test for paired samples with denoted by *. Representative flasks are shown above corresponding graph bars.

    Article Snippet: All truncated ORF2p fragments were expressed in HeLa cells following transient transfection with respective expression constructs as detected via western blot analysis with anti-Gal4 antibodies (Santa Cruz) (Figure ).

    Techniques: Generated, Sequencing, Expressing, Plasmid Preparation, Transfection, Standard Deviation

    IRE1 activation increases the surface expression of α1(A322D) subunit of GABA A receptors. ( A ) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or XBP1-s (spliced XBP1) plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. The cell lysates are then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B C ) (n = 5 for α1 and n = 3 for BiP, paired t-test). ( D ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface protein expression levels of α1 is shown in ( E ) (n = 5, paired t-test). ( F ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or XBP-s or transfected with XBP-s and treated with lactacystin (2.5 μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( G ) (n = 3, one-way ANOVA followed by Fisher test, *, p

    Journal: PLoS ONE

    Article Title: Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors

    doi: 10.1371/journal.pone.0207948

    Figure Lengend Snippet: IRE1 activation increases the surface expression of α1(A322D) subunit of GABA A receptors. ( A ) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or XBP1-s (spliced XBP1) plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. The cell lysates are then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B C ) (n = 5 for α1 and n = 3 for BiP, paired t-test). ( D ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface protein expression levels of α1 is shown in ( E ) (n = 5, paired t-test). ( F ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or XBP-s or transfected with XBP-s and treated with lactacystin (2.5 μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( G ) (n = 3, one-way ANOVA followed by Fisher test, *, p

    Article Snippet: Cell lines that stably expressing α1β2γ2 and α1(A322D)β2γ2 receptors were generated by transient transfection with α1:β2:γ2 (1:1:1) and α1(A322D):β2:γ2 (1:1:1) plasmids.

    Techniques: Activation Assay, Expressing, Transfection, SDS Page, Western Blot, Affinity Purification, Quantitation Assay

    ATF6 activation promotes the forward trafficking of α1(A322D) subunit of GABA A receptors. (A) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or HA-tagged full-length ATF6α plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Endo H resistant v1 subunit bands (top arrow, lane 4) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 3 and 4) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B ) and ( C ) (n = 5 for α1 and n = 4 for BiP, paired t-test). Quantification of the ratio of endo H resistant α1 / total α1 is shown in ( D ) (n = 3, paired t-test). ( E ) Cells were treated as in ( A ). Forty-eight hrs post transfection, the nuclear fractions were extracted and subject to SDS-PAGE. ATF6 (N) is the cleaved, activated N-terminal ATF6 in the nucleus. Matrin-3 serves as a nuclear protein loading control. ( F ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels is shown in ( G ) (n = 6, paired t-test). ( H ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or ATF6, or transfected with ATF6 and treated with lactacystin (2.5μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( I ) (n = 5, one-way ANOVA followed by Fisher test, *, p

    Journal: PLoS ONE

    Article Title: Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors

    doi: 10.1371/journal.pone.0207948

    Figure Lengend Snippet: ATF6 activation promotes the forward trafficking of α1(A322D) subunit of GABA A receptors. (A) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or HA-tagged full-length ATF6α plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Endo H resistant v1 subunit bands (top arrow, lane 4) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 3 and 4) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B ) and ( C ) (n = 5 for α1 and n = 4 for BiP, paired t-test). Quantification of the ratio of endo H resistant α1 / total α1 is shown in ( D ) (n = 3, paired t-test). ( E ) Cells were treated as in ( A ). Forty-eight hrs post transfection, the nuclear fractions were extracted and subject to SDS-PAGE. ATF6 (N) is the cleaved, activated N-terminal ATF6 in the nucleus. Matrin-3 serves as a nuclear protein loading control. ( F ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels is shown in ( G ) (n = 6, paired t-test). ( H ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or ATF6, or transfected with ATF6 and treated with lactacystin (2.5μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( I ) (n = 5, one-way ANOVA followed by Fisher test, *, p

    Article Snippet: Cell lines that stably expressing α1β2γ2 and α1(A322D)β2γ2 receptors were generated by transient transfection with α1:β2:γ2 (1:1:1) and α1(A322D):β2:γ2 (1:1:1) plasmids.

    Techniques: Activation Assay, Expressing, Transfection, Incubation, SDS Page, Western Blot, Affinity Purification, Quantitation Assay

    Cx43 forward trafficking is modulated by Cx43-20 K in PRA and PRB Cells. Representative images of human myometrial cells hTERT-HM A/B transfected with Cx43-GFP ( a,b ) or Cx43-GFP + mCh-Cx43-20 K ( c–h ) or Cx43-ML-GFP ( i,j ) or Cx43-ML-GFP + mCh-Cx43-20 K ( k–p ) and induced for PRA ( a,c,d,e,i,k,l,m ) or PRB ( b,f,g,h,j,n,o,p ) expression for 24 h and stimulated with P4 (100 nM) for 2 h. Results showed that Cx43 forward trafficking is restricted in the PRB cells transfected with Cx43-GFP ( b ) however it was rescued by co-transfection of Cx43-GFP and mCh-Cx43-20 K ( f,g,h ). The lower panel shows that the forward trafficking of Cx43 is blocked in both PRA and PRB cells when transfected with Cx43-ML-GFP (which is unable to express Cx43-20 K, i,j ), while it is rescued when Cx43-ML-GFP is co-transfected with mCh-Cx43-20 K ( k–p ). GFP is represented with green fluorescence, mCh-Cx43-20 K with red and their co-localization with yellow colour. Scale bar = 20 μm.

    Journal: Scientific Reports

    Article Title: Progesterone Via its Type-A Receptor Promotes Myometrial Gap Junction Coupling

    doi: 10.1038/s41598-017-13488-9

    Figure Lengend Snippet: Cx43 forward trafficking is modulated by Cx43-20 K in PRA and PRB Cells. Representative images of human myometrial cells hTERT-HM A/B transfected with Cx43-GFP ( a,b ) or Cx43-GFP + mCh-Cx43-20 K ( c–h ) or Cx43-ML-GFP ( i,j ) or Cx43-ML-GFP + mCh-Cx43-20 K ( k–p ) and induced for PRA ( a,c,d,e,i,k,l,m ) or PRB ( b,f,g,h,j,n,o,p ) expression for 24 h and stimulated with P4 (100 nM) for 2 h. Results showed that Cx43 forward trafficking is restricted in the PRB cells transfected with Cx43-GFP ( b ) however it was rescued by co-transfection of Cx43-GFP and mCh-Cx43-20 K ( f,g,h ). The lower panel shows that the forward trafficking of Cx43 is blocked in both PRA and PRB cells when transfected with Cx43-ML-GFP (which is unable to express Cx43-20 K, i,j ), while it is rescued when Cx43-ML-GFP is co-transfected with mCh-Cx43-20 K ( k–p ). GFP is represented with green fluorescence, mCh-Cx43-20 K with red and their co-localization with yellow colour. Scale bar = 20 μm.

    Article Snippet: Transient transfection Transient transfection was performed using TransIT-LT1 transfection reagent (Mirus Bio, USA) following manufacturer’s protocol.

    Techniques: Transfection, Expressing, Cotransfection, Fluorescence

    LMP2A augments activation of the TOPFlash luciferase reporter by β-catenin. (A) 293 cells were transfected with SV-β-galactosidase and either TOPFlash or FOPFlash plus vector, β-catenin, LMP2A, or β-catenin with LMP2A. TOPFlash contains four consensus TCF-4 binding sites in a promoter driving expression of the luciferase gene, and FOPFlash is an analogous construct with the four TCF-4 sites mutated. Transfections and assays were performed in triplicate in two separate experiments, and luciferase activity was normalized to β-galactosidase activity to control for transfection efficiency. The data are presented graphically as TOPFlash activity normalized to FOPFlash activity for each of the indicated conditions. (B) Immunoblot analyses were performed on the lysates used in the luciferase assay described above. Antibodies against HA confirm the presence of transfected HA-tagged LMP2A. Transfected and endogenous β-catenin levels were revealed with an anti-β-catenin antibody, and immunoblot with an antibody against TCF-4 attested to equal levels of this transcription factor in the transfected 293 cells.

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus Latent Membrane Protein 2A Activates ?-Catenin Signaling in Epithelial Cells

    doi: 10.1128/JVI.77.22.12276-12284.2003

    Figure Lengend Snippet: LMP2A augments activation of the TOPFlash luciferase reporter by β-catenin. (A) 293 cells were transfected with SV-β-galactosidase and either TOPFlash or FOPFlash plus vector, β-catenin, LMP2A, or β-catenin with LMP2A. TOPFlash contains four consensus TCF-4 binding sites in a promoter driving expression of the luciferase gene, and FOPFlash is an analogous construct with the four TCF-4 sites mutated. Transfections and assays were performed in triplicate in two separate experiments, and luciferase activity was normalized to β-galactosidase activity to control for transfection efficiency. The data are presented graphically as TOPFlash activity normalized to FOPFlash activity for each of the indicated conditions. (B) Immunoblot analyses were performed on the lysates used in the luciferase assay described above. Antibodies against HA confirm the presence of transfected HA-tagged LMP2A. Transfected and endogenous β-catenin levels were revealed with an anti-β-catenin antibody, and immunoblot with an antibody against TCF-4 attested to equal levels of this transcription factor in the transfected 293 cells.

    Article Snippet: In addition, in these stable cells, the level of LMP2A expression was much lower than that observed in transient transfections, and LMP2A protein was not detected in the nuclear fraction of these cells.

    Techniques: Activation Assay, Luciferase, Transfection, Plasmid Preparation, Binding Assay, Expressing, Construct, Activity Assay

    Methylation-sensitive binding of CTCF to the first exon of hTERT . (A) In vivo binding of CTCF to the first exon of hTERT in telomerase-positive and negative cell lines was analyzed by ChIP assay using anti-CTCF antibody. PCR coamplification of the test fragments ( hTERT and H19) using as template DNA input fraction and DNA recovered from immunoprecipitated fractions bound by the anti-CTCF antibody. (B) 5′-end-labeled control (F1) and SssI methylase-treated (F1-met) fragments were digested with methylation-sensitive BstUI and analyzed on polyacrylamide gels to verify efficiency of in vitro methylation. ( C ) Control unmethylated (F1) or SssI-methylated (F1-met) fragments were analyzed by gel-shift assay (EMSA). F, free probe; B, CTCF-bound probe. ( D ) Representation of the hTERT sequence cloned into the pTERT-297/ex2/FRT. Arrows represent the localization of the primers used for hTERT methylation analysis of stable transfectant. ( E ) The methylation status of the stable transfectants was verified by MS-SSCA. Unmethylated and fully methylated controls were obtained from plasmids used for stable transfection. UT and MT represent, respectively, the unmethylated and methylated plasmids stably transfected into HeLa cells, and after 30 population doublings. ( F ) Binding of CTCF to the first exon of hTERT in stably transfected cell line was analyzed by ChIP assay using anti-CTCF antibody.

    Journal: Nucleic Acids Research

    Article Title: Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene

    doi: 10.1093/nar/gkl1125

    Figure Lengend Snippet: Methylation-sensitive binding of CTCF to the first exon of hTERT . (A) In vivo binding of CTCF to the first exon of hTERT in telomerase-positive and negative cell lines was analyzed by ChIP assay using anti-CTCF antibody. PCR coamplification of the test fragments ( hTERT and H19) using as template DNA input fraction and DNA recovered from immunoprecipitated fractions bound by the anti-CTCF antibody. (B) 5′-end-labeled control (F1) and SssI methylase-treated (F1-met) fragments were digested with methylation-sensitive BstUI and analyzed on polyacrylamide gels to verify efficiency of in vitro methylation. ( C ) Control unmethylated (F1) or SssI-methylated (F1-met) fragments were analyzed by gel-shift assay (EMSA). F, free probe; B, CTCF-bound probe. ( D ) Representation of the hTERT sequence cloned into the pTERT-297/ex2/FRT. Arrows represent the localization of the primers used for hTERT methylation analysis of stable transfectant. ( E ) The methylation status of the stable transfectants was verified by MS-SSCA. Unmethylated and fully methylated controls were obtained from plasmids used for stable transfection. UT and MT represent, respectively, the unmethylated and methylated plasmids stably transfected into HeLa cells, and after 30 population doublings. ( F ) Binding of CTCF to the first exon of hTERT in stably transfected cell line was analyzed by ChIP assay using anti-CTCF antibody.

    Article Snippet: Transient transfection of reporter plasmids (0.75 μg/well) was carried out in triplicate using JetPEI Cationic Polymer Transfection reagent (4 μl/well) (Polyplus-transfection, Illkirch, France).

    Techniques: Methylation, Binding Assay, In Vivo, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation, Labeling, In Vitro, Electrophoretic Mobility Shift Assay, Sequencing, Clone Assay, Transfection, Mass Spectrometry, Stable Transfection

    Inhibition of encystation by AcAtg3-siRNA. Cells transfected with Superfect Transfection reagent, nonspecific negative siRNA, or AcAtg3-siRNA were incubated in encystment media for 3 days and the number of mature cysts scored. The formation of mature cysts was almost inhibited in AcAtg3-siRNA transfected cells.

    Journal: The Korean Journal of Parasitology

    Article Title: Atg3-Mediated Lipidation of Atg8 Is Involved in Encystation of Acanthamoeba

    doi: 10.3347/kjp.2011.49.2.103

    Figure Lengend Snippet: Inhibition of encystation by AcAtg3-siRNA. Cells transfected with Superfect Transfection reagent, nonspecific negative siRNA, or AcAtg3-siRNA were incubated in encystment media for 3 days and the number of mature cysts scored. The formation of mature cysts was almost inhibited in AcAtg3-siRNA transfected cells.

    Article Snippet: Transient transfection was performed by Superfect transfection reagent (Qiagen, Valencia, California, USA) as previously described [ ].

    Techniques: Inhibition, Transfection, Incubation

    Transfection of pUbAcAtg3g for the intracellular localization of Atg3. Expressed Atg3-EGFP showed dispersed fluorescence in the cytoplasm in the trophozoites (encystation-0 hr) and the early stage of encystation (encystation-20 hr). After incubation for 40 hr in encystment media, AcAtg3-EGFP localized around autophagosomal membrane (encystation-40 hr).

    Journal: The Korean Journal of Parasitology

    Article Title: Atg3-Mediated Lipidation of Atg8 Is Involved in Encystation of Acanthamoeba

    doi: 10.3347/kjp.2011.49.2.103

    Figure Lengend Snippet: Transfection of pUbAcAtg3g for the intracellular localization of Atg3. Expressed Atg3-EGFP showed dispersed fluorescence in the cytoplasm in the trophozoites (encystation-0 hr) and the early stage of encystation (encystation-20 hr). After incubation for 40 hr in encystment media, AcAtg3-EGFP localized around autophagosomal membrane (encystation-40 hr).

    Article Snippet: Transient transfection was performed by Superfect transfection reagent (Qiagen, Valencia, California, USA) as previously described [ ].

    Techniques: Transfection, Fluorescence, Incubation

    Inhibition of Atg8-PE conjugation by AcAtg3-siRNA. The formation of the Atg8-PE conjugate was inhibited by AcAtg3-siRNA transfection. Lane 1; trophozoite, lane 2; encysting cells for 1 day, lane 3; encysting cells for 2 days, lane 4; encysting cells for 1 day after transfection with AcAtg3-siRNA, and lane 5; encysting cells for 2 days after transfection with AcAtg3-siRNA.

    Journal: The Korean Journal of Parasitology

    Article Title: Atg3-Mediated Lipidation of Atg8 Is Involved in Encystation of Acanthamoeba

    doi: 10.3347/kjp.2011.49.2.103

    Figure Lengend Snippet: Inhibition of Atg8-PE conjugation by AcAtg3-siRNA. The formation of the Atg8-PE conjugate was inhibited by AcAtg3-siRNA transfection. Lane 1; trophozoite, lane 2; encysting cells for 1 day, lane 3; encysting cells for 2 days, lane 4; encysting cells for 1 day after transfection with AcAtg3-siRNA, and lane 5; encysting cells for 2 days after transfection with AcAtg3-siRNA.

    Article Snippet: Transient transfection was performed by Superfect transfection reagent (Qiagen, Valencia, California, USA) as previously described [ ].

    Techniques: Inhibition, Conjugation Assay, Transfection