Structured Review

Promega mgcl2
Autoradiographs showing cleavage of 50mer fragments containing GT, GU, IT or IU mismatches. The position of the cleavage product is indicated by the arrow. The reaction was performed in 10 mM HEPES/NaOH pH 7.5 containing 100 mM NaCl and 10 mM <t>MgCl2</t> . Samples were removed from the reaction at various times after adding the enzyme and stopped by adding to formamide containing 10 mM EDTA, as described in Materials and Methods. The reaction time (min) is shown at the top of each gel lane. These digests were performed in the absence of BSA and did not proceed to completion.
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Related Products / Commonly Used Together

atpase
nadh-coupled
kapa taq polymerase
dntp
kapa taq buffer

Images

1) Product Images from "Recognition of GT mismatches by Vsr mismatch endonuclease"

Article Title: Recognition of GT mismatches by Vsr mismatch endonuclease

Journal:

doi:

Autoradiographs showing cleavage of 50mer fragments containing GT, GU, IT or IU mismatches. The position of the cleavage product is indicated by the arrow. The reaction was performed in 10 mM HEPES/NaOH pH 7.5 containing 100 mM NaCl and 10 mM MgCl2 . Samples were removed from the reaction at various times after adding the enzyme and stopped by adding to formamide containing 10 mM EDTA, as described in Materials and Methods. The reaction time (min) is shown at the top of each gel lane. These digests were performed in the absence of BSA and did not proceed to completion.
Figure Legend Snippet: Autoradiographs showing cleavage of 50mer fragments containing GT, GU, IT or IU mismatches. The position of the cleavage product is indicated by the arrow. The reaction was performed in 10 mM HEPES/NaOH pH 7.5 containing 100 mM NaCl and 10 mM MgCl2 . Samples were removed from the reaction at various times after adding the enzyme and stopped by adding to formamide containing 10 mM EDTA, as described in Materials and Methods. The reaction time (min) is shown at the top of each gel lane. These digests were performed in the absence of BSA and did not proceed to completion.

Techniques Used:

2) Product Images from "Pentamidine inhibits catalytic activity of group I intron Ca.LSU by altering RNA folding"

Article Title: Pentamidine inhibits catalytic activity of group I intron Ca.LSU by altering RNA folding

Journal:

doi:

Preincubation of Ca.LSU precursor with pentamidine enhances its inhibitory effect on splicing. Equal amounts of radiolabeled Ca.LSU precursor RNA were added to each reaction. The non-preincubated reactions were run under standard splicing conditions with the indicated concentrations of pentamidine (lanes 1–5). The other samples were preincubated in Tris buffer at 37°C for 20 min before addition of MgCl2 , spermidine and GTP. The indicated concentrations of pentamidine were added either during preincubation (lanes 6–10) or after preincubation together with the other splicing components (lanes 11–16) and reaction products were analyzed by PAGE as described in Materials and Methods. The asterisks indicate products possibly generated by Mg2+ -induced hydrolysis of the Ca.LSU precursor RNA.
Figure Legend Snippet: Preincubation of Ca.LSU precursor with pentamidine enhances its inhibitory effect on splicing. Equal amounts of radiolabeled Ca.LSU precursor RNA were added to each reaction. The non-preincubated reactions were run under standard splicing conditions with the indicated concentrations of pentamidine (lanes 1–5). The other samples were preincubated in Tris buffer at 37°C for 20 min before addition of MgCl2 , spermidine and GTP. The indicated concentrations of pentamidine were added either during preincubation (lanes 6–10) or after preincubation together with the other splicing components (lanes 11–16) and reaction products were analyzed by PAGE as described in Materials and Methods. The asterisks indicate products possibly generated by Mg2+ -induced hydrolysis of the Ca.LSU precursor RNA.

Techniques Used: Polyacrylamide Gel Electrophoresis, Generated

3) Product Images from "Folding of the group I intron ribozyme from the 26S rRNA gene of Candida albicans"

Article Title: Folding of the group I intron ribozyme from the 26S rRNA gene of Candida albicans

Journal:

doi:

Effect of cations on Ca.LSU folding assayed by ribonuclease T1 cleavage. The 5′-end-labeled Ca.LSU intron was assayed as in Figure 2, except that the indicated concentrations of cations were added to each sample together with 50 mM Tris–HCl pH 7.5. ( A , opposite) A representative gel, showing the effects of MgCl2 , CaCl2 and spermidine present during incubation prior to ribonuclease T1 digestion. Lane (–), undigested RNA. The 0°C lane represents RNA preincubated at that temperature rather than at 37°C, as were all other samples. ( B , above) Effect of addition of MgCl2 or CaCl2 on ribonuclease T1 cleavage. This panel is represented as in Figure 2B, except orange symbols indicate sites at which ribonuclease T1 cleavage is reduced by incubation with these cations. Orange Gs, complete protection; orange arrows and dots, the reduced level of cleavage observed in the presence of these cations; black arrows and letters, no change relative to incubation in buffer alone; blue dots, sites at which ribonuclease T1 did not cleave Ca.LSU incubated in buffer alone, but at which cleavage was induced by incubation with these cations. ( C , above) Spermidine effect on ribonuclease T1 cleavage. Green symbols, differences in the T1 cleavage pattern in the presence of spermidine as opposed to the pattern in the presence of MgCl2 or CaCl2 (B); orange letters, arrows and dots, sites at which the effect of spermidine was the same as that of MgCl2 or CaCl2 ; blue dots, sites at which ribonuclease T1 did not cleave Ca.LSU incubated in buffer alone, but at which cleavage was equivalently induced by incubation with divalent metal ions or spermidine.
Figure Legend Snippet: Effect of cations on Ca.LSU folding assayed by ribonuclease T1 cleavage. The 5′-end-labeled Ca.LSU intron was assayed as in Figure 2, except that the indicated concentrations of cations were added to each sample together with 50 mM Tris–HCl pH 7.5. ( A , opposite) A representative gel, showing the effects of MgCl2 , CaCl2 and spermidine present during incubation prior to ribonuclease T1 digestion. Lane (–), undigested RNA. The 0°C lane represents RNA preincubated at that temperature rather than at 37°C, as were all other samples. ( B , above) Effect of addition of MgCl2 or CaCl2 on ribonuclease T1 cleavage. This panel is represented as in Figure 2B, except orange symbols indicate sites at which ribonuclease T1 cleavage is reduced by incubation with these cations. Orange Gs, complete protection; orange arrows and dots, the reduced level of cleavage observed in the presence of these cations; black arrows and letters, no change relative to incubation in buffer alone; blue dots, sites at which ribonuclease T1 did not cleave Ca.LSU incubated in buffer alone, but at which cleavage was induced by incubation with these cations. ( C , above) Spermidine effect on ribonuclease T1 cleavage. Green symbols, differences in the T1 cleavage pattern in the presence of spermidine as opposed to the pattern in the presence of MgCl2 or CaCl2 (B); orange letters, arrows and dots, sites at which the effect of spermidine was the same as that of MgCl2 or CaCl2 ; blue dots, sites at which ribonuclease T1 did not cleave Ca.LSU incubated in buffer alone, but at which cleavage was equivalently induced by incubation with divalent metal ions or spermidine.

Techniques Used: Labeling, Incubation

Presence of oligovalent cations during preincubation prevents activation of Ca.LSU self-splicing. ( A ) Equal amounts of radiolabeled Ca.LSU precursor were preincubated in Tris–HCl pH 7.5 in the presence of 1.25 mM MgCl2 , 0.4 mM spermidine, and 10 µM GTP, or with no additions (control) at 37°C for 5 min. Then the missing splicing components were added to standard concentrations, and MgCl2 , spermidine and GTP were brought to standard splicing concentrations. Splicing reactions were run at 37°C for the indicated times. The splicing products were analyzed as in Figure 4B. ( B ) Equal amounts of radiolabeled precursor RNA were preincubated in Tris–HCl pH 7.5 in the presence of the indicated concentration of each cation at 37°C for 20 min, and then the splicing components were added to each sample to the standard concentrations except for 1.25 mM MgCl2 and 1.25 mM CaCl2 for all CaCl2 samples, and 2.5 mM MgCl2 for MgCl2 and spermidine samples; this change was necessary to maintain the same divalent cation concentration in all splicing reactions in each series of experiments. ( C ) Equal amounts of radiolabeled Ca.LSU precursor RNA were diluted in 10 µl of 50 mM Tris–HCl pH 7.5 with the indicated concentrations of MgCl2 , and were incubated at the indicated temperatures for 20 min before analysis by native polyacrylamide gel electrophoresis.
Figure Legend Snippet: Presence of oligovalent cations during preincubation prevents activation of Ca.LSU self-splicing. ( A ) Equal amounts of radiolabeled Ca.LSU precursor were preincubated in Tris–HCl pH 7.5 in the presence of 1.25 mM MgCl2 , 0.4 mM spermidine, and 10 µM GTP, or with no additions (control) at 37°C for 5 min. Then the missing splicing components were added to standard concentrations, and MgCl2 , spermidine and GTP were brought to standard splicing concentrations. Splicing reactions were run at 37°C for the indicated times. The splicing products were analyzed as in Figure 4B. ( B ) Equal amounts of radiolabeled precursor RNA were preincubated in Tris–HCl pH 7.5 in the presence of the indicated concentration of each cation at 37°C for 20 min, and then the splicing components were added to each sample to the standard concentrations except for 1.25 mM MgCl2 and 1.25 mM CaCl2 for all CaCl2 samples, and 2.5 mM MgCl2 for MgCl2 and spermidine samples; this change was necessary to maintain the same divalent cation concentration in all splicing reactions in each series of experiments. ( C ) Equal amounts of radiolabeled Ca.LSU precursor RNA were diluted in 10 µl of 50 mM Tris–HCl pH 7.5 with the indicated concentrations of MgCl2 , and were incubated at the indicated temperatures for 20 min before analysis by native polyacrylamide gel electrophoresis.

Techniques Used: Activation Assay, Concentration Assay, Incubation, Polyacrylamide Gel Electrophoresis

Related Articles

Clone Assay:

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Centrifugation:

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Amplification:

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Article Title: Pentamidine inhibits catalytic activity of group I intron Ca.LSU by altering RNA folding
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Synthesized:

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Autoradiography:

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Construct:

Article Title: Toxic Activity, Molecular Modeling and Docking Simulations of Bacillus thuringiensis Cry11 Toxin Variants Obtained via DNA Shuffling
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Article Title: Toxic Activity, Molecular Modeling and Docking Simulations of Bacillus thuringiensis Cry11 Toxin Variants Obtained via DNA Shuffling
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Electrophoresis:

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Article Title: Optimization of genetic transformation of Artemisia annua L. Using Agrobacterium for Artemisinin production
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Random Hexamer Labeling:

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Formalin-fixed Paraffin-Embedded:

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Activity Assay:

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Expressing:

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Modification:

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Article Title: Population genetic signatures of a climate change driven marine range extension
Article Snippet: Each PCR contained 4.725 µL of double distilled H2 0, 6.25 µL of MyTaq Redmix (Bioline), 0.075 µL of 10 mM forward primer, 0.25 µL of 10 mM reverse primer, 0.20 µL of 5pmol/µL fluorophore labelled primer, and 1 µL (18–37 ng) of DNA. .. PCR conditions were modified slightly to optimize PCR products for some samples, such that 1 µL of 25 mM MgCl2 (Promega) was added in place of water. .. The number of cycles was reduced from 35 to 30, and the final extension was reduced from 5 to 3 min. Capillary separation of PCR products was performed by the Australian Genome Research Facility Ltd (AGRF).

Transformation Assay:

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Reverse Transcription Polymerase Chain Reaction:

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Article Title: A Direct Effect of Sex Hormones on Epithelial Barrier Function in Inflammatory Bowel Disease Models
Article Snippet: Paragraph title: 2.3. Reverse Transcriptase Polymerase Chain Reaction (rt-PCR) ... PCR was performed in a 25 μL reaction, using GoTaq polymerase and GoTaq Flexi buffer, 2 mM MgCl2 (Promega, Madison, WI, USA), dNTP (0.5 mM each, Roche, Basel, Switzerland), 50 ng template and 0.5 nM primer (for primers sequences see ).

Polymerase Chain Reaction:

Article Title: Site History and Edaphic Features Override the Influence of Plant Species on Microbial Communities in Restored Tidal Freshwater Wetlands
Article Snippet: Each reaction mixture contained 0.20 μM forward and reverse primers, 0.20 mM dNTPs, 1.75 mM MgCl2 , 1× GoTaq colorless flexi buffer (Promega Corporation, Madison, WI) with 1.5 mM MgCl2 , 0.064% BSA, and 0.025 Taq U/μl GoTaq hot-start polymerase (Promega Corporation, Madison, WI). .. Each reaction mixture contained 0.20 μM forward and reverse primers, 0.20 mM dNTPs, 1.75 mM MgCl2 , 1× GoTaq colorless flexi buffer (Promega Corporation, Madison, WI) with 1.5 mM MgCl2 , 0.064% BSA, and 0.025 Taq U/μl GoTaq hot-start polymerase (Promega Corporation, Madison, WI).

Article Title: Pentamidine inhibits catalytic activity of group I intron Ca.LSU by altering RNA folding
Article Snippet: The PCR amplified DNA fragment containing the SP6 promoter at the 5′ end of the sense strand of a fragment of the rRNA gene containing Ca.LSU was transcribed in vitro in the presence of 500 µM rATP, rCTP and rGTP, 200 µM rUTP (Promega) and 20 µCi [α-32 P]UTP (3000 Ci/mmol; NEN-DuPont), as previously described ( ). .. The standard self-splicing assay was performed in 10 µl reactions containing 50 mM Tris–HCl (pH 7.5), 1.25 mM MgCl2 , 0.4 mM spermidine, 10 µM GTP and 10 U RNasin (Promega) at 37°C for 20 min. Where indicated, precursor RNA was preincubated in 8 µl of 62.5 mM Tris–HCl (pH 7.5) plus 10 U RNasin with the indicated components at 37°C for the indicated times prior to initiating the self-splicing reaction by addition of 2 µl of reaction mix containing all reaction components at concentrations to bring each to the standard assay conditions.

Article Title: Inhibition of IL-17 and IL-23 in Human Keratinocytes by the A3 Adenosine Receptor Agonist Piclidenoson
Article Snippet: Tissue sections on slides were deparaffinized in xylene and rehydrated by washing in serial dilutions of ethanol. .. After rehydration, 20 μ l of solution A [1.25X PCR buffer (200 mM Tris–HCl and 500 mM KCl), 6.25 mM MgCl2, 5 units RNasin (Promega, Madison, WI, USA), 2 mM DTT, and 1 unit RQ1 RNase-free DNase (Promega)] was applied directly to the marked area. .. The psoriatic skin lesion and the adjacent tissue were completely scraped off the slide using a pipette tip and were collected to a different microcentrifuge tube.

Article Title: Identification in milk of a serum amyloid A peptide chemoattractant for B lymphoblasts
Article Snippet: PCR amplification was performed using the Pfu DNA polymerase (Promega France, Charbonnières, France). .. PCR was carried out for SAA cDNA amplification, in a total volume of 20 μl containing 100 ng of cDNA, 1 U Taq polymerase (Promega France, Charbonnières, France), 0.2 mM of each dNTP, 0.5 μM of each primers, 1× Taq polymerase buffer (Promega France, Charbonnières, France), 2 mM MgCl2 (Promega France, Charbonnières, France). .. Sense F1 (5'- TCTGACATGAGAGAGGCCAACTAC-3') and anti-sense R1 (5'-GTCAGGCAGGCCACGAGGTCT-3') primers were designed on the basis of our porcine SAA cDNA sequence obtained from the F0-R0 PCR product.

Article Title: Toxic Activity, Molecular Modeling and Docking Simulations of Bacillus thuringiensis Cry11 Toxin Variants Obtained via DNA Shuffling
Article Snippet: Paragraph title: Isolation of cry11 Genes via PCR ... Briefly, reactions were conducted in a final volume of 50 μl that contained 20 ng of plasmid DNA, 0.5 μM primers, 1× Taq polymerase buffer, 0.4 mM dNTPs, 1.5 mM MgCl2 and 0.5 U GoTaq polymerase (Promega).

Article Title: Optimization of genetic transformation of Artemisia annua L. Using Agrobacterium for Artemisinin production
Article Snippet: The forward primer was 5’-AGTGGCAGTGAAGGGCGAACAGT-3’ and reverse 5’- AATAACGGTTCAGGCAC AGCACA-3’, designed according to the sequence of gus gene. .. The 25 mL of PCR reactions included 0.5 mM forward primer, 0.5 mM reverse primer, 2.5 μl 10 × Taq DNA Polymerase buffer, 0.2 mM dNTPs, 1 U Taq DNA Polymerase, and 2 mM MgCl2 (Promega). .. Amplification was performed in a thermal cycler (Applied Biosystem - 2720 Thermal cycler) as follows: 1 min at 94°C; 30 cycles of 30 s at 94°C; 30 s at 72°C; followed by 7 min at 72°C.

Article Title: Anti-Melanogenic Effect of Oenothera laciniata Methanol Extract in Melan-a Cells
Article Snippet: Total RNA (5 μg) was reverse transcribed using 8 μL of Molony murine leukemia virus RT (M-MLV RT) 5 × buffer, 3 μL of 10 mM dNTPs, 0.45 μL of 40 U/μL RNasein inhibitor, 0.3 μL of 200 U/μL M-MLV RT (Promega, Madison, WI, USA), and 1.5 μL of 50 μM oligo dT (Bioneer, Daejeon, Korea) in a 40 μL volume. .. Single-stranded cDNA was then amplified by PCR using 4 μL of 5 × green Go Taq flexi buffer, 0.4 μL of 10 mM dNTPs, 0.1 μL of 5 U/μL Taq polymerase, 1.2 μL of 25 mM MgCl2 (Promega), and 0.4 μL of 20 μM of specific sense and anti-sense primers of tyrosinase, TRP-1, TRP-2, MITF-M, or β-actin. .. The primer sequences used for PCR were as follows: tyrosinase forward 5′-CAT TTT TGA TTT GAG TGT CT-3′, reverse 5′-TGT GGT AGT CGT CTT TGT CC-3′; TRP-1 forward 5′-GCT GCA GGA GCC TTC TTT CTC-3′, reverse 5′-AAG ACG CTG CAC TGC TGG TCT-3′; TRP-2 forward 5′-GGA TGA CCG TGA GCA ATG GCC-3′, reverse 5′-CGG TTG TGA CCA ATG GGT GCC-3′; MITF-M forward 5′-TAC AGA AAG TAG AGG GAG GAG GAC TAA G-3′, reverse 5′-CAC AGT TGG AGT TAA GAG TGA GCA TAG CC-3′; β-actin forward 5′-ACC GTG AAA AGA TGA CCC AG-3′, reverse 5′-TAC GGA TGT CAA CGT CAC AC-3′.

Article Title: A Direct Effect of Sex Hormones on Epithelial Barrier Function in Inflammatory Bowel Disease Models
Article Snippet: RNA was isolated using a NucleoSpin® RNA kit (MACHEREY-NAGEL, Düren, Germany) and cDNA was synthesized using the TAKARA reverse transcription system (TAKARA BIO INC, Shiga, Japan). .. PCR was performed in a 25 μL reaction, using GoTaq polymerase and GoTaq Flexi buffer, 2 mM MgCl2 (Promega, Madison, WI, USA), dNTP (0.5 mM each, Roche, Basel, Switzerland), 50 ng template and 0.5 nM primer (for primers sequences see ). .. Quantitative PCR (QPCR) was used to determine claudin 1 , claudin 2 , zonula occludens 1 (ZO-1 ) and occludin expression.

Article Title: Gene expression pattern of TCR repertoire and alteration expression of IL-17A gene of γδ T cells in patients with acute myocardial infarction
Article Snippet: Aliquots of the cDNA (1 μL) were amplified in 20 μL reactions using one of the three Vγ primers and a Cγ primer or one of the eight Vδ primers and a Cδ primer. .. The final reaction mixture contained 0.5 μM of the sense and antisense primers, 1× PCR buffer, 0.1 mM dNTPs, 1.5 mM MgCl2 , and 1.25 U Taq polymerase (Promega, CA, USA). .. The amplification was performed on a DNA thermal cycler (BioMetra, Germany).

Article Title: Molecular Characterization of Clostridium botulinum Isolates from Foodborne Outbreaks in Thailand, 2010
Article Snippet: The method of Umeda et al. was used for PCR typing of boNT /A, and Umeda et al. for boNT /B typing except that 1 U of GoTaq ® DNA polymerase and its associated buffer were used (Promega, Madison, WI, USA). .. The PCR method was described by Umeda et al. using 1 U of GoTaq ® DNA polymerase and 1.5 mM MgCl2 (Promega, Madison, WI, USA). .. The amplified products of the ha33 gene and p47 gene confirmed as C. botulinum type A1 strain and type A2 strain, respectively.

Article Title: Molecular Characterization of Clostridium botulinum Isolates from Foodborne Outbreaks in Thailand, 2010
Article Snippet: DNA was eluted with 100 µl elution buffer and stored at 4°C until use. .. The PCR was modified from the method of Lindström et al. and used to identify BoNT types A through F. A 30 µl of reaction mix containing 0.5 µl of extracted DNA template, 0.1 µM of each primer, 200 nM of each deoxynucleotide triphosphate (dNTP), 5× Buffer, 1 U of GoTaq ® DNA polymerase and 1.5 mM MgCl2 (Promega, Madison, WI, USA) was used. .. PCR was for 35 cycles, including a denaturation step at 95°C for 20 sec, annealing at 55°C for 30 sec and extension at 62°C for 2 min.

Article Title: Population genetic signatures of a climate change driven marine range extension
Article Snippet: Each PCR contained 4.725 µL of double distilled H2 0, 6.25 µL of MyTaq Redmix (Bioline), 0.075 µL of 10 mM forward primer, 0.25 µL of 10 mM reverse primer, 0.20 µL of 5pmol/µL fluorophore labelled primer, and 1 µL (18–37 ng) of DNA. .. PCR conditions were modified slightly to optimize PCR products for some samples, such that 1 µL of 25 mM MgCl2 (Promega) was added in place of water. .. The number of cycles was reduced from 35 to 30, and the final extension was reduced from 5 to 3 min. Capillary separation of PCR products was performed by the Australian Genome Research Facility Ltd (AGRF).

Multiplexing:

Article Title: Site History and Edaphic Features Override the Influence of Plant Species on Microbial Communities in Restored Tidal Freshwater Wetlands
Article Snippet: Multiplexing and sequencing of all 57 samples was accomplished using a 10-bp MIDS barcoded F515 primer also containing a Roche 454-A pyrosequencing adaptor (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3′; Roche Applied Science, Branford, CT, USA) and a GA linker sequence. .. Each reaction mixture contained 0.20 μM forward and reverse primers, 0.20 mM dNTPs, 1.75 mM MgCl2 , 1× GoTaq colorless flexi buffer (Promega Corporation, Madison, WI) with 1.5 mM MgCl2 , 0.064% BSA, and 0.025 Taq U/μl GoTaq hot-start polymerase (Promega Corporation, Madison, WI).

Fluorescence:

Article Title: Gene expression pattern of TCR repertoire and alteration expression of IL-17A gene of γδ T cells in patients with acute myocardial infarction
Article Snippet: The final reaction mixture contained 0.5 μM of the sense and antisense primers, 1× PCR buffer, 0.1 mM dNTPs, 1.5 mM MgCl2 , and 1.25 U Taq polymerase (Promega, CA, USA). .. Aliquots of the unlabeled PCR products (2 μL) were subjected to a cycle of runoff reaction using fluorophore-labeled Cγ-FAM or Cδ-FAM primer respectively.

Isolation:

Article Title: Toxic Activity, Molecular Modeling and Docking Simulations of Bacillus thuringiensis Cry11 Toxin Variants Obtained via DNA Shuffling
Article Snippet: Paragraph title: Isolation of cry11 Genes via PCR ... Briefly, reactions were conducted in a final volume of 50 μl that contained 20 ng of plasmid DNA, 0.5 μM primers, 1× Taq polymerase buffer, 0.4 mM dNTPs, 1.5 mM MgCl2 and 0.5 U GoTaq polymerase (Promega).

Article Title: Anti-Melanogenic Effect of Oenothera laciniata Methanol Extract in Melan-a Cells
Article Snippet: Total RNA was isolated using Trizol-Reagent (Invitrogen, New York, NY, USA) according to the manufacturer’s instructions. .. Single-stranded cDNA was then amplified by PCR using 4 μL of 5 × green Go Taq flexi buffer, 0.4 μL of 10 mM dNTPs, 0.1 μL of 5 U/μL Taq polymerase, 1.2 μL of 25 mM MgCl2 (Promega), and 0.4 μL of 20 μM of specific sense and anti-sense primers of tyrosinase, TRP-1, TRP-2, MITF-M, or β-actin.

Article Title: A Direct Effect of Sex Hormones on Epithelial Barrier Function in Inflammatory Bowel Disease Models
Article Snippet: RNA was isolated using a NucleoSpin® RNA kit (MACHEREY-NAGEL, Düren, Germany) and cDNA was synthesized using the TAKARA reverse transcription system (TAKARA BIO INC, Shiga, Japan). .. PCR was performed in a 25 μL reaction, using GoTaq polymerase and GoTaq Flexi buffer, 2 mM MgCl2 (Promega, Madison, WI, USA), dNTP (0.5 mM each, Roche, Basel, Switzerland), 50 ng template and 0.5 nM primer (for primers sequences see ).

Size-exclusion Chromatography:

Article Title: Anti-Melanogenic Effect of Oenothera laciniata Methanol Extract in Melan-a Cells
Article Snippet: Single-stranded cDNA was then amplified by PCR using 4 μL of 5 × green Go Taq flexi buffer, 0.4 μL of 10 mM dNTPs, 0.1 μL of 5 U/μL Taq polymerase, 1.2 μL of 25 mM MgCl2 (Promega), and 0.4 μL of 20 μM of specific sense and anti-sense primers of tyrosinase, TRP-1, TRP-2, MITF-M, or β-actin. .. The expected sizes of the PCR products of tyrosinase, TRP-1, TRP-2, MITF-M, and β-actin, respectively, were 1192, 268, 1044, 326, and 528 base pairs.

Labeling:

Article Title: Pentamidine inhibits catalytic activity of group I intron Ca.LSU by altering RNA folding
Article Snippet: Paragraph title: Labeling and self-splicing of Ca.LSU precursor ... The standard self-splicing assay was performed in 10 µl reactions containing 50 mM Tris–HCl (pH 7.5), 1.25 mM MgCl2 , 0.4 mM spermidine, 10 µM GTP and 10 U RNasin (Promega) at 37°C for 20 min. Where indicated, precursor RNA was preincubated in 8 µl of 62.5 mM Tris–HCl (pH 7.5) plus 10 U RNasin with the indicated components at 37°C for the indicated times prior to initiating the self-splicing reaction by addition of 2 µl of reaction mix containing all reaction components at concentrations to bring each to the standard assay conditions.

Article Title: Gene expression pattern of TCR repertoire and alteration expression of IL-17A gene of γδ T cells in patients with acute myocardial infarction
Article Snippet: The final reaction mixture contained 0.5 μM of the sense and antisense primers, 1× PCR buffer, 0.1 mM dNTPs, 1.5 mM MgCl2 , and 1.25 U Taq polymerase (Promega, CA, USA). .. Aliquots of the unlabeled PCR products (2 μL) were subjected to a cycle of runoff reaction using fluorophore-labeled Cγ-FAM or Cδ-FAM primer respectively.

Purification:

Article Title: Catalysts from synthetic genetic polymers
Article Snippet: Purified single-stranded libraries and substrates were annealed by incubation at 80°C for 60 s, then allowed to cool to room temperature over 5 min, except for phosphorylimidazolide oligos, which were not annealed. .. For selection of RNA ligase and endonuclease XNAzymes, reactions were performed at 17°C in 30 mM HEPES (pH 8.5), 150 mM KCl, 25mM MgCl2 and 0.5 U/ul RNasein RNase inhibitor (Promega, USA).

Article Title: Pentamidine inhibits catalytic activity of group I intron Ca.LSU by altering RNA folding
Article Snippet: The transcribed precursor RNA was purified by PAGE on a 5% polyacrylamide–8 M urea gel ( , ). .. The standard self-splicing assay was performed in 10 µl reactions containing 50 mM Tris–HCl (pH 7.5), 1.25 mM MgCl2 , 0.4 mM spermidine, 10 µM GTP and 10 U RNasin (Promega) at 37°C for 20 min. Where indicated, precursor RNA was preincubated in 8 µl of 62.5 mM Tris–HCl (pH 7.5) plus 10 U RNasin with the indicated components at 37°C for the indicated times prior to initiating the self-splicing reaction by addition of 2 µl of reaction mix containing all reaction components at concentrations to bring each to the standard assay conditions.

Article Title: A Mechanism for the Activation of the Influenza Virus Transcriptase
Article Snippet: Synthetic RNAs 5′-ppAAUCUAUAAUAGCAUUAUCC-3′ and 5′-ppGAAUACUCAAG-3′ (Chemgenes) were capped and radiolabelled by incubation with [α-32 P] GTP, vaccinia virus capping enzyme (NEB) and 2′-O -methyltransferase (NEB), following the manufacturer’s instructions. .. For endonuclease activity assays, 0.5 μL capped RNA was mixed with 1.5 μL purified polymerase (at a final concentration of 142 nM) in 7.5 mM MgCl2 , 2 mM DTT, and 1 U/μl RNasin (Promega), with or without 0.5 μM 5′ vRNA promoter (5′-AGCAGUAGCAAGGGG-3′) and 0.5 μM 3′ vRNA promoter (5′-CCCCUGCUUCUGCU-3′), in a total reaction volume of 4 μl. .. For capped RNA primed transcription assays, ATP, UTP, CTP and GTP were added at a final concentration of 0.5 mM each.

Article Title: microRNAs stimulate translation initiation mediated by HCV-like IRESes
Article Snippet: A total of 3.105 electroporated HeLa cells were lysed in 100 μl of RLNa buffer (10 mM Tris–HCl (pH 8), 10 mM NaCl, 3 mM MgCl2 , 1 mM DTT, 0.5% NP40 and 10 U/ml of RNaseIN (Promega Co., Madison, WI, USA). .. A total of 3.105 electroporated HeLa cells were lysed in 100 μl of RLNa buffer (10 mM Tris–HCl (pH 8), 10 mM NaCl, 3 mM MgCl2 , 1 mM DTT, 0.5% NP40 and 10 U/ml of RNaseIN (Promega Co., Madison, WI, USA).

Sequencing:

Article Title: Site History and Edaphic Features Override the Influence of Plant Species on Microbial Communities in Restored Tidal Freshwater Wetlands
Article Snippet: Multiplexing and sequencing of all 57 samples was accomplished using a 10-bp MIDS barcoded F515 primer also containing a Roche 454-A pyrosequencing adaptor (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3′; Roche Applied Science, Branford, CT, USA) and a GA linker sequence. .. Each reaction mixture contained 0.20 μM forward and reverse primers, 0.20 mM dNTPs, 1.75 mM MgCl2 , 1× GoTaq colorless flexi buffer (Promega Corporation, Madison, WI) with 1.5 mM MgCl2 , 0.064% BSA, and 0.025 Taq U/μl GoTaq hot-start polymerase (Promega Corporation, Madison, WI).

Article Title: Identification in milk of a serum amyloid A peptide chemoattractant for B lymphoblasts
Article Snippet: As no homologous porcine EST was identified based on the porcine SAA cDNA sequence, primers F0 et R0 were designed from dog, mink, guinea pig, rabbit and human SAA cDNA sequences, CM59173, in a region relatively well conserved between SAA of different subtypes and different animal origins, using VectorNTI software (Invitrogen, Cergy Pontoise, France). .. PCR was carried out for SAA cDNA amplification, in a total volume of 20 μl containing 100 ng of cDNA, 1 U Taq polymerase (Promega France, Charbonnières, France), 0.2 mM of each dNTP, 0.5 μM of each primers, 1× Taq polymerase buffer (Promega France, Charbonnières, France), 2 mM MgCl2 (Promega France, Charbonnières, France).

Article Title: Optimization of genetic transformation of Artemisia annua L. Using Agrobacterium for Artemisinin production
Article Snippet: The forward primer was 5’-AGTGGCAGTGAAGGGCGAACAGT-3’ and reverse 5’- AATAACGGTTCAGGCAC AGCACA-3’, designed according to the sequence of gus gene. .. The 25 mL of PCR reactions included 0.5 mM forward primer, 0.5 mM reverse primer, 2.5 μl 10 × Taq DNA Polymerase buffer, 0.2 mM dNTPs, 1 U Taq DNA Polymerase, and 2 mM MgCl2 (Promega).

Selection:

Article Title: Catalysts from synthetic genetic polymers
Article Snippet: Purified single-stranded libraries and substrates were annealed by incubation at 80°C for 60 s, then allowed to cool to room temperature over 5 min, except for phosphorylimidazolide oligos, which were not annealed. .. For selection of RNA ligase and endonuclease XNAzymes, reactions were performed at 17°C in 30 mM HEPES (pH 8.5), 150 mM KCl, 25mM MgCl2 and 0.5 U/ul RNasein RNase inhibitor (Promega, USA). .. For selection of XNA (FANA) ligase XNAzymes, reactions were performed at 35°C in 30 mM HEPES (pH 7.2), 150 mM KCl, 25mM MgCl2 and 1mM ZnCl2 .

Polyacrylamide Gel Electrophoresis:

Article Title: Pentamidine inhibits catalytic activity of group I intron Ca.LSU by altering RNA folding
Article Snippet: The transcribed precursor RNA was purified by PAGE on a 5% polyacrylamide–8 M urea gel ( , ). .. The standard self-splicing assay was performed in 10 µl reactions containing 50 mM Tris–HCl (pH 7.5), 1.25 mM MgCl2 , 0.4 mM spermidine, 10 µM GTP and 10 U RNasin (Promega) at 37°C for 20 min. Where indicated, precursor RNA was preincubated in 8 µl of 62.5 mM Tris–HCl (pH 7.5) plus 10 U RNasin with the indicated components at 37°C for the indicated times prior to initiating the self-splicing reaction by addition of 2 µl of reaction mix containing all reaction components at concentrations to bring each to the standard assay conditions.

Article Title: A Mechanism for the Activation of the Influenza Virus Transcriptase
Article Snippet: For endonuclease activity assays, 0.5 μL capped RNA was mixed with 1.5 μL purified polymerase (at a final concentration of 142 nM) in 7.5 mM MgCl2 , 2 mM DTT, and 1 U/μl RNasin (Promega), with or without 0.5 μM 5′ vRNA promoter (5′-AGCAGUAGCAAGGGG-3′) and 0.5 μM 3′ vRNA promoter (5′-CCCCUGCUUCUGCU-3′), in a total reaction volume of 4 μl. .. Pol II CTD peptides ( ) were added at a final concentration of 1.42 μM where indicated.

Plasmid Preparation:

Article Title: Toxic Activity, Molecular Modeling and Docking Simulations of Bacillus thuringiensis Cry11 Toxin Variants Obtained via DNA Shuffling
Article Snippet: The cry11 genes were amplified via PCR using specific primers and plasmid DNA from constructs pBTM3, pSV2 and pJEG90.1 as templates. .. Briefly, reactions were conducted in a final volume of 50 μl that contained 20 ng of plasmid DNA, 0.5 μM primers, 1× Taq polymerase buffer, 0.4 mM dNTPs, 1.5 mM MgCl2 and 0.5 U GoTaq polymerase (Promega). .. The PCR products were separated via electrophoresis and purified using the PCR Clean-Up System (Promega).

Article Title: Optimization of genetic transformation of Artemisia annua L. Using Agrobacterium for Artemisinin production
Article Snippet: [ ] The transformant cells of Agrobacterium harboring the binary vector pCAMBIA were confirmed by crude polymerase chain reaction (PCR) and electrophoresis. .. The 25 mL of PCR reactions included 0.5 mM forward primer, 0.5 mM reverse primer, 2.5 μl 10 × Taq DNA Polymerase buffer, 0.2 mM dNTPs, 1 U Taq DNA Polymerase, and 2 mM MgCl2 (Promega).

Article Title: Toxic Activity, Molecular Modeling and Docking Simulations of Bacillus thuringiensis Cry11 Toxin Variants Obtained via DNA Shuffling
Article Snippet: The cry11 genes were amplified via PCR using specific primers and plasmid DNA from constructs pBTM3, pSV2 and pJEG90.1 as templates. .. Briefly, reactions were conducted in a final volume of 50 μl that contained 20 ng of plasmid DNA, 0.5 μM primers, 1× Taq polymerase buffer, 0.4 mM dNTPs, 1.5 mM MgCl2 and 0.5 U Go Taq polymerase (Promega). .. The PCR products were separated via electrophoresis and purified using the PCR Clean-Up System (Promega).

Software:

Article Title: Identification in milk of a serum amyloid A peptide chemoattractant for B lymphoblasts
Article Snippet: As no homologous porcine EST was identified based on the porcine SAA cDNA sequence, primers F0 et R0 were designed from dog, mink, guinea pig, rabbit and human SAA cDNA sequences, CM59173, in a region relatively well conserved between SAA of different subtypes and different animal origins, using VectorNTI software (Invitrogen, Cergy Pontoise, France). .. PCR was carried out for SAA cDNA amplification, in a total volume of 20 μl containing 100 ng of cDNA, 1 U Taq polymerase (Promega France, Charbonnières, France), 0.2 mM of each dNTP, 0.5 μM of each primers, 1× Taq polymerase buffer (Promega France, Charbonnières, France), 2 mM MgCl2 (Promega France, Charbonnières, France).

Article Title: Gene expression pattern of TCR repertoire and alteration expression of IL-17A gene of γδ T cells in patients with acute myocardial infarction
Article Snippet: The final reaction mixture contained 0.5 μM of the sense and antisense primers, 1× PCR buffer, 0.1 mM dNTPs, 1.5 mM MgCl2 , and 1.25 U Taq polymerase (Promega, CA, USA). .. Aliquots of the unlabeled PCR products (2 μL) were subjected to a cycle of runoff reaction using fluorophore-labeled Cγ-FAM or Cδ-FAM primer respectively.

Multiplex Assay:

Article Title: Molecular Characterization of Clostridium botulinum Isolates from Foodborne Outbreaks in Thailand, 2010
Article Snippet: Paragraph title: Multiplex PCR of ha33 and p47genes for Confirmation of boNT/ A and boNT /B Typing ... The PCR method was described by Umeda et al. using 1 U of GoTaq ® DNA polymerase and 1.5 mM MgCl2 (Promega, Madison, WI, USA).

Positron Emission Tomography:

Article Title: Population genetic signatures of a climate change driven marine range extension
Article Snippet: Microsatellite loci were assigned unique fluorophores (FAM, VIC, NED, PET) to enable fluorescent tagging of PCR products. .. PCR conditions were modified slightly to optimize PCR products for some samples, such that 1 µL of 25 mM MgCl2 (Promega) was added in place of water.

Agarose Gel Electrophoresis:

Article Title: Identification in milk of a serum amyloid A peptide chemoattractant for B lymphoblasts
Article Snippet: PCR was carried out for SAA cDNA amplification, in a total volume of 20 μl containing 100 ng of cDNA, 1 U Taq polymerase (Promega France, Charbonnières, France), 0.2 mM of each dNTP, 0.5 μM of each primers, 1× Taq polymerase buffer (Promega France, Charbonnières, France), 2 mM MgCl2 (Promega France, Charbonnières, France). .. PCR was carried out for SAA cDNA amplification, in a total volume of 20 μl containing 100 ng of cDNA, 1 U Taq polymerase (Promega France, Charbonnières, France), 0.2 mM of each dNTP, 0.5 μM of each primers, 1× Taq polymerase buffer (Promega France, Charbonnières, France), 2 mM MgCl2 (Promega France, Charbonnières, France).

Article Title: Optimization of genetic transformation of Artemisia annua L. Using Agrobacterium for Artemisinin production
Article Snippet: The 25 mL of PCR reactions included 0.5 mM forward primer, 0.5 mM reverse primer, 2.5 μl 10 × Taq DNA Polymerase buffer, 0.2 mM dNTPs, 1 U Taq DNA Polymerase, and 2 mM MgCl2 (Promega). .. Amplification was performed in a thermal cycler (Applied Biosystem - 2720 Thermal cycler) as follows: 1 min at 94°C; 30 cycles of 30 s at 94°C; 30 s at 72°C; followed by 7 min at 72°C.

Article Title: Anti-Melanogenic Effect of Oenothera laciniata Methanol Extract in Melan-a Cells
Article Snippet: Single-stranded cDNA was then amplified by PCR using 4 μL of 5 × green Go Taq flexi buffer, 0.4 μL of 10 mM dNTPs, 0.1 μL of 5 U/μL Taq polymerase, 1.2 μL of 25 mM MgCl2 (Promega), and 0.4 μL of 20 μM of specific sense and anti-sense primers of tyrosinase, TRP-1, TRP-2, MITF-M, or β-actin. .. The PCR conditions were as follows: tyrosinase and TRP-1, 28 cycles of denaturation at 94°C for 60 sec, annealing at 56°C for 60 sec, and extension at 72°C for 60 sec; TRP-2, 28 cycles of denaturation at 94°C for 60 sec, annealing at 64°C for 60 sec, and extension at 72°C for 60 sec; MITF-M, 30 cycles of denaturation at 94°C for 30 sec, annealing at 54°C for 30 sec, and extension at 72°C for 30 sec; β-actin, 30 cycles of denaturation at 94°C for 30 sec, annealing at 51°C for 30 sec, and extension at 72°C for 60 sec.

In Vitro:

Article Title: Pentamidine inhibits catalytic activity of group I intron Ca.LSU by altering RNA folding
Article Snippet: The PCR amplified DNA fragment containing the SP6 promoter at the 5′ end of the sense strand of a fragment of the rRNA gene containing Ca.LSU was transcribed in vitro in the presence of 500 µM rATP, rCTP and rGTP, 200 µM rUTP (Promega) and 20 µCi [α-32 P]UTP (3000 Ci/mmol; NEN-DuPont), as previously described ( ). .. The standard self-splicing assay was performed in 10 µl reactions containing 50 mM Tris–HCl (pH 7.5), 1.25 mM MgCl2 , 0.4 mM spermidine, 10 µM GTP and 10 U RNasin (Promega) at 37°C for 20 min. Where indicated, precursor RNA was preincubated in 8 µl of 62.5 mM Tris–HCl (pH 7.5) plus 10 U RNasin with the indicated components at 37°C for the indicated times prior to initiating the self-splicing reaction by addition of 2 µl of reaction mix containing all reaction components at concentrations to bring each to the standard assay conditions.

Article Title: A Mechanism for the Activation of the Influenza Virus Transcriptase
Article Snippet: Paragraph title: In Vitro FluPol Activity Assays ... For endonuclease activity assays, 0.5 μL capped RNA was mixed with 1.5 μL purified polymerase (at a final concentration of 142 nM) in 7.5 mM MgCl2 , 2 mM DTT, and 1 U/μl RNasin (Promega), with or without 0.5 μM 5′ vRNA promoter (5′-AGCAGUAGCAAGGGG-3′) and 0.5 μM 3′ vRNA promoter (5′-CCCCUGCUUCUGCU-3′), in a total reaction volume of 4 μl.

Incubation:

Article Title: Catalysts from synthetic genetic polymers
Article Snippet: Purified single-stranded libraries and substrates were annealed by incubation at 80°C for 60 s, then allowed to cool to room temperature over 5 min, except for phosphorylimidazolide oligos, which were not annealed. .. For selection of RNA ligase and endonuclease XNAzymes, reactions were performed at 17°C in 30 mM HEPES (pH 8.5), 150 mM KCl, 25mM MgCl2 and 0.5 U/ul RNasein RNase inhibitor (Promega, USA).

Article Title: Inhibition of IL-17 and IL-23 in Human Keratinocytes by the A3 Adenosine Receptor Agonist Piclidenoson
Article Snippet: After rehydration, 20 μ l of solution A [1.25X PCR buffer (200 mM Tris–HCl and 500 mM KCl), 6.25 mM MgCl2, 5 units RNasin (Promega, Madison, WI, USA), 2 mM DTT, and 1 unit RQ1 RNase-free DNase (Promega)] was applied directly to the marked area. .. The samples were treated with proteinase K at a final concentration of 0.1 mg/ml.

Article Title: A Mechanism for the Activation of the Influenza Virus Transcriptase
Article Snippet: Synthetic RNAs 5′-ppAAUCUAUAAUAGCAUUAUCC-3′ and 5′-ppGAAUACUCAAG-3′ (Chemgenes) were capped and radiolabelled by incubation with [α-32 P] GTP, vaccinia virus capping enzyme (NEB) and 2′-O -methyltransferase (NEB), following the manufacturer’s instructions. .. For endonuclease activity assays, 0.5 μL capped RNA was mixed with 1.5 μL purified polymerase (at a final concentration of 142 nM) in 7.5 mM MgCl2 , 2 mM DTT, and 1 U/μl RNasin (Promega), with or without 0.5 μM 5′ vRNA promoter (5′-AGCAGUAGCAAGGGG-3′) and 0.5 μM 3′ vRNA promoter (5′-CCCCUGCUUCUGCU-3′), in a total reaction volume of 4 μl.

DNA Extraction:

Article Title: Population genetic signatures of a climate change driven marine range extension
Article Snippet: Paragraph title: DNA extraction, PCR amplification and genotyping ... PCR conditions were modified slightly to optimize PCR products for some samples, such that 1 µL of 25 mM MgCl2 (Promega) was added in place of water.

Concentration Assay:

Article Title: Folding of the group I intron ribozyme from the 26S rRNA gene of Candida albicans
Article Snippet: The splicing reaction of uniformly 32 P-labeled precursor containing Ca.LSU was assayed in 10 µl reactions in the presence of 50 mM Tris–HCl pH 7.5, 1.25 mM MgCl2 , 0.4 mM spermidine, 10 µM GTP and 10 U of RNasin (Promega) at 37°C for 20 min unless otherwise indicated. .. Prior to the reaction, preincubation (where indicated) was performed in 8 µl in the presence of precursor RNA, indicated concentrations of pentamidine, cations or GTP at 37°C for the indicated period of time.

Article Title: Inhibition of IL-17 and IL-23 in Human Keratinocytes by the A3 Adenosine Receptor Agonist Piclidenoson
Article Snippet: After rehydration, 20 μ l of solution A [1.25X PCR buffer (200 mM Tris–HCl and 500 mM KCl), 6.25 mM MgCl2, 5 units RNasin (Promega, Madison, WI, USA), 2 mM DTT, and 1 unit RQ1 RNase-free DNase (Promega)] was applied directly to the marked area. .. The psoriatic skin lesion and the adjacent tissue were completely scraped off the slide using a pipette tip and were collected to a different microcentrifuge tube.

Article Title: A Mechanism for the Activation of the Influenza Virus Transcriptase
Article Snippet: Synthetic RNAs 5′-ppAAUCUAUAAUAGCAUUAUCC-3′ and 5′-ppGAAUACUCAAG-3′ (Chemgenes) were capped and radiolabelled by incubation with [α-32 P] GTP, vaccinia virus capping enzyme (NEB) and 2′-O -methyltransferase (NEB), following the manufacturer’s instructions. .. For endonuclease activity assays, 0.5 μL capped RNA was mixed with 1.5 μL purified polymerase (at a final concentration of 142 nM) in 7.5 mM MgCl2 , 2 mM DTT, and 1 U/μl RNasin (Promega), with or without 0.5 μM 5′ vRNA promoter (5′-AGCAGUAGCAAGGGG-3′) and 0.5 μM 3′ vRNA promoter (5′-CCCCUGCUUCUGCU-3′), in a total reaction volume of 4 μl. .. For capped RNA primed transcription assays, ATP, UTP, CTP and GTP were added at a final concentration of 0.5 mM each.

Fractionation:

Article Title: Huntingtin mediates dendritic transport of ?-actin mRNA in rat neurons
Article Snippet: Paragraph title: Glycerol gradient fractionation of mouse brain lysate ... One wild-type post-natal day 15 mouse brain was homogenized in 2 ml of buffer containing 10 mM HEPES, pH 7.6, 1.5 mM MgCl2 , protease inhibitors, and 40 units RNAsin (Promega) in a dounce homogenizer with 6 strokes pestle A and 10 strokes pestle B.

Staining:

Article Title: Inhibition of IL-17 and IL-23 in Human Keratinocytes by the A3 Adenosine Receptor Agonist Piclidenoson
Article Snippet: After rehydration, 20 μ l of solution A [1.25X PCR buffer (200 mM Tris–HCl and 500 mM KCl), 6.25 mM MgCl2, 5 units RNasin (Promega, Madison, WI, USA), 2 mM DTT, and 1 unit RQ1 RNase-free DNase (Promega)] was applied directly to the marked area. .. After rehydration, 20 μ l of solution A [1.25X PCR buffer (200 mM Tris–HCl and 500 mM KCl), 6.25 mM MgCl2, 5 units RNasin (Promega, Madison, WI, USA), 2 mM DTT, and 1 unit RQ1 RNase-free DNase (Promega)] was applied directly to the marked area.

Article Title: Identification in milk of a serum amyloid A peptide chemoattractant for B lymphoblasts
Article Snippet: PCR was carried out for SAA cDNA amplification, in a total volume of 20 μl containing 100 ng of cDNA, 1 U Taq polymerase (Promega France, Charbonnières, France), 0.2 mM of each dNTP, 0.5 μM of each primers, 1× Taq polymerase buffer (Promega France, Charbonnières, France), 2 mM MgCl2 (Promega France, Charbonnières, France). .. PCR was carried out for SAA cDNA amplification, in a total volume of 20 μl containing 100 ng of cDNA, 1 U Taq polymerase (Promega France, Charbonnières, France), 0.2 mM of each dNTP, 0.5 μM of each primers, 1× Taq polymerase buffer (Promega France, Charbonnières, France), 2 mM MgCl2 (Promega France, Charbonnières, France).

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  • 81
    Promega gsk3β activity reaction buffer
    12B2 and 15C2 are specific for nonphospho-S GSK3 in brain lysates of human, mouse, and rat, and both effectively immunoprecipitate GSK3 from cell lysates. (A) Protein sequence alignments for <t>GSK3β</t> (amino acids 1–25) and GSK3α (amino acids 13–37) from human, mouse and rat (Uniprot IDs in parentheses). (B,C) Blots of lysates from human, mouse and rat cortical tissue and GAPDH was used as a loading control (40 μg/lane total protein loaded; experiment repeated three times). (B) 12B2 (red) specifically labeled GSK3β, not GSK3α, in lysates and total GSK3α/β (green) was used to identify both isoforms. (C) 15C2 (red) labeled both GSK3β and GSK3α in lysates and total GSK3α/β (green) was used to identify both isoforms. (D–F) The 12B2 (D) , 15C2 (E) , or control mouse IgG ( F , Ms IgG) were used to immunoprecipitate GSK3 enzymes from HEK293T cell lysates. The starting lysate (Input) was incubated with magnetic beads coated with 12B2 (D) , 15C2 (E) , or Ms IgG control (F) antibodies. 12B2 pulled down only GSK3β (12B2-IP), 15C2 pulled down both GSK3α and β (15C2-IP) and Ms IgG did not pull down GSK3α or β (MsIgG-IP). The post-IP lysates were also run for comparisons to the input samples. These experiments were performed three independent times.
    Gsk3β Activity Reaction Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 81/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 81 stars, based on 1 article reviews
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    gsk3β activity reaction buffer - by Bioz Stars, 2019-12
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    79
    Promega atp based celltiter glo luminescent cell viability assay solution
    Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using <t>Caspase-Glo</t> 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic <t>ATP</t> levels were measured using ATP-based <t>CellTiter-Glo</t> Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P
    Atp Based Celltiter Glo Luminescent Cell Viability Assay Solution, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega pcr mix
    Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase <t>PCR,</t> using primers from 5 BLV genome regions, was used to amplify products from <t>DNA</t> extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.
    Pcr Mix, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Promega atp glo assay
    Activation of MEK/ERK signalling by hypoactive BRAF mutants is RAS-dependent a , V5-tagged wild-type (WT) or mutant BRAF kinases were expressed in 293H cells that stably express NRAS(Q61K). These BRAF protein kinases were isolated with anti-V5 agarose. The in vitro . b . c , In vitro kinase activity of the indicated BRAF proteins which were isolated from 293H cells that stably express NRAS(Q61K) was assessed as described in a . d . e , NIH3T3 cells expressing the indicated BRAF proteins were stimulated with 100 ng ml −1 . f were cultured in doxycycline (30 ng ml −1 . g . h , The frequency of coexistent RAS or NF1 alterations in human BRAF mutant melanomas compared to that in NSCLC and colorectal cancers.. The calculation was based on the sample sets as shown in . The P values were calculated by using a paired t -test. N.S., not significant. i , The phosphorylation of multiple RTKs in the indicated cell lines was assayed using the Human Phospho-RTK Array Kit. Phosphorylated RTKs are highlighted with boxes in different colours. j . k , Cells were cultured and exposed to cetuximab at concentrations of 0, 0.3, 1, 3, 10, 30, 100 and 300 nM for 3 days. The effects of drug on cell growth were quantified using the <t>ATP-Glo</t> assay. Graphs were generated using Prism 6 (mean ±s.d. are represented by the dots and error bars, n =8). l . m , The effects of cetuximab or trametinib on the growth of the cells described in l was determined by ATP-Glo assay after 3 days treatment. Graphs were generated using Prism 6 (mean ± s.d., n = 8). n . o , Growth inhibition of the cells described in n after three days exposure to varying doses of cetuximab on day 3 was determined by ATP-Glo assay. Graphs were generated using Prism 6 (mean ± s.d., n =8) Fig. 1d
    Atp Glo Assay, supplied by Promega, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    12B2 and 15C2 are specific for nonphospho-S GSK3 in brain lysates of human, mouse, and rat, and both effectively immunoprecipitate GSK3 from cell lysates. (A) Protein sequence alignments for GSK3β (amino acids 1–25) and GSK3α (amino acids 13–37) from human, mouse and rat (Uniprot IDs in parentheses). (B,C) Blots of lysates from human, mouse and rat cortical tissue and GAPDH was used as a loading control (40 μg/lane total protein loaded; experiment repeated three times). (B) 12B2 (red) specifically labeled GSK3β, not GSK3α, in lysates and total GSK3α/β (green) was used to identify both isoforms. (C) 15C2 (red) labeled both GSK3β and GSK3α in lysates and total GSK3α/β (green) was used to identify both isoforms. (D–F) The 12B2 (D) , 15C2 (E) , or control mouse IgG ( F , Ms IgG) were used to immunoprecipitate GSK3 enzymes from HEK293T cell lysates. The starting lysate (Input) was incubated with magnetic beads coated with 12B2 (D) , 15C2 (E) , or Ms IgG control (F) antibodies. 12B2 pulled down only GSK3β (12B2-IP), 15C2 pulled down both GSK3α and β (15C2-IP) and Ms IgG did not pull down GSK3α or β (MsIgG-IP). The post-IP lysates were also run for comparisons to the input samples. These experiments were performed three independent times.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: 12B2 and 15C2 are specific for nonphospho-S GSK3 in brain lysates of human, mouse, and rat, and both effectively immunoprecipitate GSK3 from cell lysates. (A) Protein sequence alignments for GSK3β (amino acids 1–25) and GSK3α (amino acids 13–37) from human, mouse and rat (Uniprot IDs in parentheses). (B,C) Blots of lysates from human, mouse and rat cortical tissue and GAPDH was used as a loading control (40 μg/lane total protein loaded; experiment repeated three times). (B) 12B2 (red) specifically labeled GSK3β, not GSK3α, in lysates and total GSK3α/β (green) was used to identify both isoforms. (C) 15C2 (red) labeled both GSK3β and GSK3α in lysates and total GSK3α/β (green) was used to identify both isoforms. (D–F) The 12B2 (D) , 15C2 (E) , or control mouse IgG ( F , Ms IgG) were used to immunoprecipitate GSK3 enzymes from HEK293T cell lysates. The starting lysate (Input) was incubated with magnetic beads coated with 12B2 (D) , 15C2 (E) , or Ms IgG control (F) antibodies. 12B2 pulled down only GSK3β (12B2-IP), 15C2 pulled down both GSK3α and β (15C2-IP) and Ms IgG did not pull down GSK3α or β (MsIgG-IP). The post-IP lysates were also run for comparisons to the input samples. These experiments were performed three independent times.

    Article Snippet: After sample incubation, the wells were rinsed and incubated in 50 μl/well GSK3β activity reaction buffer [100 μM ATP (V915A, Promega), 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2 and 0.1 mg/ml bovine serum albumin] for 1 h at 30°C.

    Techniques: Sequencing, Labeling, Mass Spectrometry, Incubation, Magnetic Beads

    Treating cells with protein phosphatase inhibitor decreases npS9 GSK3β in cells. (A) A standard curve of dephosphorylated GSK3β protein captured with 12B2 antibody was used for quantitative sandwich ELISAs ( r 2 = 0.999). (B) Untreated HEK293T lysates assayed in 12B2 sandwich ELISAs at 120, 60, 30, 15, and 7.5 μg total protein/well produces a linear dose response curve ( r 2 = 0.988). Interpolation using the standard curve in (A) indicates that the lysate samples contain 7.4, 5.2, 3.5, 2.0, and 0.9 ng of npS9 GSK3β, respectively. (C) HEK293T cells were either untreated (-) or treated with 10 nM calyculin A for 30 min (+) to reduce npS9 GSK3β levels ( n = 4 independent experiments). The lysates were used in 12B2 sandwich ELISAs. A significant reduction in npS9 GSK3β levels was detected in calyculin A (10 nM) treated cells compared to untreated cells ( ∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: Treating cells with protein phosphatase inhibitor decreases npS9 GSK3β in cells. (A) A standard curve of dephosphorylated GSK3β protein captured with 12B2 antibody was used for quantitative sandwich ELISAs ( r 2 = 0.999). (B) Untreated HEK293T lysates assayed in 12B2 sandwich ELISAs at 120, 60, 30, 15, and 7.5 μg total protein/well produces a linear dose response curve ( r 2 = 0.988). Interpolation using the standard curve in (A) indicates that the lysate samples contain 7.4, 5.2, 3.5, 2.0, and 0.9 ng of npS9 GSK3β, respectively. (C) HEK293T cells were either untreated (-) or treated with 10 nM calyculin A for 30 min (+) to reduce npS9 GSK3β levels ( n = 4 independent experiments). The lysates were used in 12B2 sandwich ELISAs. A significant reduction in npS9 GSK3β levels was detected in calyculin A (10 nM) treated cells compared to untreated cells ( ∗ p

    Article Snippet: After sample incubation, the wells were rinsed and incubated in 50 μl/well GSK3β activity reaction buffer [100 μM ATP (V915A, Promega), 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2 and 0.1 mg/ml bovine serum albumin] for 1 h at 30°C.

    Techniques:

    Protein phosphatases regulate GSK3β phosphorylation independent of Akt signaling. HEK293T cells were treated with an Akt inhibitor (AZD-5363, 1 μM), a protein phosphatase inhibitor (calyculin A, 10 nM) or the Akt inhibitor followed by the phosphatase inhibitor. Four independent experiments were run. (A) Western blots of samples were probed with 12B2 (npS9-GSK3β specific), total GSK3α/β, pS9-GSK3β and GAPDH (loading control). (B) Quantitation of the blots shows that inhibition of Akt (AZD) significantly increased npS9 GSK3β, while inhibition of protein phosphatases (Caly) significantly reduced npS9 GSK3β. When Akt signaling was blocked first and then the phosphatase inhibitor was applied (AZD + Caly) a significant reduction in the level of npS9 GSK3β occurred when compared to Akt inhibitor alone. (C) Quantitation of the pS9 GSK3β blots shows an opposite pattern where inhibition of Akt significantly decreased pS9 GSK3β, while inhibition of phosphatases significantly increased pS9 GSK3β. When Akt signaling was blocked and then the phosphatase inhibitor was applied a significant increase in the level of pS9 GSK3β occurred when compared to Akt treatment alone. (D) Western blots of samples were probed with 15C2 (npS9-GSK3α/β specific), total GSK3α/β, pS9-GSK3β and GAPDH (loading control). (E,F) Quantitation of the blots shows that inhibition of Akt significantly increased npS9 GSK3α and β, while inhibition of protein phosphatases significantly reduced npS9 GSK3α and β. When Akt signaling was blocked and then the phosphatase inhibitor was applied a significant reduction in the level of npS9 GSK3β and npS21 GSK3α occurred when compared to Akt inhibitor alone. Collectively, these results suggest protein phosphatases dephosphorylate Ser9/21 independent of Akt signaling. All bands are normalized to GAPDH. All groups were statistically significant from the others in ( B,C,E,F) , but only ∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: Protein phosphatases regulate GSK3β phosphorylation independent of Akt signaling. HEK293T cells were treated with an Akt inhibitor (AZD-5363, 1 μM), a protein phosphatase inhibitor (calyculin A, 10 nM) or the Akt inhibitor followed by the phosphatase inhibitor. Four independent experiments were run. (A) Western blots of samples were probed with 12B2 (npS9-GSK3β specific), total GSK3α/β, pS9-GSK3β and GAPDH (loading control). (B) Quantitation of the blots shows that inhibition of Akt (AZD) significantly increased npS9 GSK3β, while inhibition of protein phosphatases (Caly) significantly reduced npS9 GSK3β. When Akt signaling was blocked first and then the phosphatase inhibitor was applied (AZD + Caly) a significant reduction in the level of npS9 GSK3β occurred when compared to Akt inhibitor alone. (C) Quantitation of the pS9 GSK3β blots shows an opposite pattern where inhibition of Akt significantly decreased pS9 GSK3β, while inhibition of phosphatases significantly increased pS9 GSK3β. When Akt signaling was blocked and then the phosphatase inhibitor was applied a significant increase in the level of pS9 GSK3β occurred when compared to Akt treatment alone. (D) Western blots of samples were probed with 15C2 (npS9-GSK3α/β specific), total GSK3α/β, pS9-GSK3β and GAPDH (loading control). (E,F) Quantitation of the blots shows that inhibition of Akt significantly increased npS9 GSK3α and β, while inhibition of protein phosphatases significantly reduced npS9 GSK3α and β. When Akt signaling was blocked and then the phosphatase inhibitor was applied a significant reduction in the level of npS9 GSK3β and npS21 GSK3α occurred when compared to Akt inhibitor alone. Collectively, these results suggest protein phosphatases dephosphorylate Ser9/21 independent of Akt signaling. All bands are normalized to GAPDH. All groups were statistically significant from the others in ( B,C,E,F) , but only ∗ p

    Article Snippet: After sample incubation, the wells were rinsed and incubated in 50 μl/well GSK3β activity reaction buffer [100 μM ATP (V915A, Promega), 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2 and 0.1 mg/ml bovine serum albumin] for 1 h at 30°C.

    Techniques: Western Blot, Quantitation Assay, Inhibition

    Protein phosphatase inhibition significantly reduces GSK3β kinase activity in cells. (A) A standard curve of active GSK3β enzyme (300 – 9.4 ng) confirmed the signal in the experimental samples was within the linear range of detection in this assay ( r 2 = 0.97). Experiment was repeated three times. (B) Calyculin A treated cells showed a significant reduction in GSK3β kinase activity compared to control cells (the –TCS sample sets; all samples were used at 60 μg total protein/well). Interpolation from the recombinant GSK3β enzyme activity curve with known amounts of active GSK3β indicated that the control samples contained 29 ng of active GSK3β and calyculin A treated cells contained 15 ng. Addition of TCS-2002 (0.1 mM; +TCS), a potent GSK3β inhibitor, completely blocked kinase activity in control and calyculin A treated cells ( ∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: Protein phosphatase inhibition significantly reduces GSK3β kinase activity in cells. (A) A standard curve of active GSK3β enzyme (300 – 9.4 ng) confirmed the signal in the experimental samples was within the linear range of detection in this assay ( r 2 = 0.97). Experiment was repeated three times. (B) Calyculin A treated cells showed a significant reduction in GSK3β kinase activity compared to control cells (the –TCS sample sets; all samples were used at 60 μg total protein/well). Interpolation from the recombinant GSK3β enzyme activity curve with known amounts of active GSK3β indicated that the control samples contained 29 ng of active GSK3β and calyculin A treated cells contained 15 ng. Addition of TCS-2002 (0.1 mM; +TCS), a potent GSK3β inhibitor, completely blocked kinase activity in control and calyculin A treated cells ( ∗ p

    Article Snippet: After sample incubation, the wells were rinsed and incubated in 50 μl/well GSK3β activity reaction buffer [100 μM ATP (V915A, Promega), 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2 and 0.1 mg/ml bovine serum albumin] for 1 h at 30°C.

    Techniques: Inhibition, Activity Assay, Recombinant

    12B2 is specific for nonphospho-S9 recombinant GSK3β, and 15C2 is specific for nonphospho-S9/21 recombinant GSK3β/α. (A–C) Western blots of recombinant GSK3β and α alone (input, In), phosphorylated S9 GSK3β and S21 GSK3α proteins (Ph; i.e., Akt1 treated) and dephosphorylated S9 GSK3β and S21 GSK3α proteins (NP; i.e., alkaline phosphatase treated). The GSK3α is GST-tagged and GSK3β is his-tagged, and in each blot the 12B2 (A) , 15C2 (B) or pS9 GSK3β (C) antibodies are red, while total GSK3α/β antibody is green. (D) Quantitation of the 12B2 blots shows strong reactivity with npS9 GSK3β, and none with npS21 GSK3α, pS9 GSK3β or pS21 GSK3α. (E) Quantitation of the 15C2 blots shows strong reactivity with npS9 GSK3β and npS21 GSK3α, but none with pS9 GSK3β or pS21 GSK3α. (F) Quantitation of the pS9 GSK3 blots confirm that the Akt1 treatment produced robust phosphorylation of S9 in GSK3β (and S21 in α) and that the alkaline phosphatase treatment removed phosphorylation of S9 in GSK3β (and S21 in α). Each experiment was repeated three-four independent times and all samples were loaded at 50 ng GSK3/lane. The data are normalized to total GSK3 signal.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: 12B2 is specific for nonphospho-S9 recombinant GSK3β, and 15C2 is specific for nonphospho-S9/21 recombinant GSK3β/α. (A–C) Western blots of recombinant GSK3β and α alone (input, In), phosphorylated S9 GSK3β and S21 GSK3α proteins (Ph; i.e., Akt1 treated) and dephosphorylated S9 GSK3β and S21 GSK3α proteins (NP; i.e., alkaline phosphatase treated). The GSK3α is GST-tagged and GSK3β is his-tagged, and in each blot the 12B2 (A) , 15C2 (B) or pS9 GSK3β (C) antibodies are red, while total GSK3α/β antibody is green. (D) Quantitation of the 12B2 blots shows strong reactivity with npS9 GSK3β, and none with npS21 GSK3α, pS9 GSK3β or pS21 GSK3α. (E) Quantitation of the 15C2 blots shows strong reactivity with npS9 GSK3β and npS21 GSK3α, but none with pS9 GSK3β or pS21 GSK3α. (F) Quantitation of the pS9 GSK3 blots confirm that the Akt1 treatment produced robust phosphorylation of S9 in GSK3β (and S21 in α) and that the alkaline phosphatase treatment removed phosphorylation of S9 in GSK3β (and S21 in α). Each experiment was repeated three-four independent times and all samples were loaded at 50 ng GSK3/lane. The data are normalized to total GSK3 signal.

    Article Snippet: After sample incubation, the wells were rinsed and incubated in 50 μl/well GSK3β activity reaction buffer [100 μM ATP (V915A, Promega), 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2 and 0.1 mg/ml bovine serum albumin] for 1 h at 30°C.

    Techniques: Recombinant, Western Blot, Quantitation Assay, Produced

    siRNA knockdown of GSK3α and GSK3β demonstrate specificity of the 12B2 antibody. (A) HEK293T cells were treated with control, GSK3α, GSK3β or GAPDH siRNAs and probed with 12B2 (red) and total GSK3β/α (green) antibodies. (B) Quantitation of 12B2 signal shows that GSK3β siRNA caused a reduction of 50% for GSK3β when compared to control cells, while GSK3α siRNA caused an increase in GSK3β (+35%). (C) Quantitation of total GSK3α/β antibody signal shows that GSK3α siRNA caused a loss of 66% for GSK3α and an increase in GSK3β (+29%) when compared to controls. Quantitation of total GSK3α/β antibody signal shows that GSK3β siRNA caused a loss of 41% for GSK3β and an increase in the GSK3α (+17%) when compared to control cells. All immunoblotting data are normalized to GAPDH signal and expressed as percent of the control group to illustrate the siRNA-mediated changes in signal. (D) Immunocytofluorescence of HEK293T cells confirms the reduction in 12B2 detection of npS9 GSK3β, which produces a punctate staining pattern, in GSK3β siRNA treated cells compared to control and GSK3α siRNA treated cells. Scale bars = 20 μm. Four independent experiments were performed. GAPDH siRNA quantitation is provided in Supplementary Figure S4 .

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: siRNA knockdown of GSK3α and GSK3β demonstrate specificity of the 12B2 antibody. (A) HEK293T cells were treated with control, GSK3α, GSK3β or GAPDH siRNAs and probed with 12B2 (red) and total GSK3β/α (green) antibodies. (B) Quantitation of 12B2 signal shows that GSK3β siRNA caused a reduction of 50% for GSK3β when compared to control cells, while GSK3α siRNA caused an increase in GSK3β (+35%). (C) Quantitation of total GSK3α/β antibody signal shows that GSK3α siRNA caused a loss of 66% for GSK3α and an increase in GSK3β (+29%) when compared to controls. Quantitation of total GSK3α/β antibody signal shows that GSK3β siRNA caused a loss of 41% for GSK3β and an increase in the GSK3α (+17%) when compared to control cells. All immunoblotting data are normalized to GAPDH signal and expressed as percent of the control group to illustrate the siRNA-mediated changes in signal. (D) Immunocytofluorescence of HEK293T cells confirms the reduction in 12B2 detection of npS9 GSK3β, which produces a punctate staining pattern, in GSK3β siRNA treated cells compared to control and GSK3α siRNA treated cells. Scale bars = 20 μm. Four independent experiments were performed. GAPDH siRNA quantitation is provided in Supplementary Figure S4 .

    Article Snippet: After sample incubation, the wells were rinsed and incubated in 50 μl/well GSK3β activity reaction buffer [100 μM ATP (V915A, Promega), 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2 and 0.1 mg/ml bovine serum albumin] for 1 h at 30°C.

    Techniques: Quantitation Assay, Staining

    GSK3β antibody immunostaining in cultured cells and tissue sections. (A,B) HEK293T cells stained with 12B2 ( A , green) and 15C2 ( B , green). ( C,D ) Undifferentiated SH-SY5Y cells stained with 12B2 ( C , green) and 15C2 ( D , green). (E,F) Rat primary cortical neurons (E18) stained with 12B2 ( E , green) and 15C2 ( F , green). In A-F , all cells were also stained with total GSK3α/β (red) and DAPI (blue in merged image). The pattern of staining with 12B2 and 15C2 was punctate staining throughout the cells, and 12B2 produced stronger signal than 15C2 in each cell type. Scale bars = 25 μm. (G,H) Brain sections in rat (left, retrosplenial cortex displayed) and human (right, temporal cortex) stained with 12B2 (G) or 15C2 (H) . In general, both antibodies produced clear somatodendritic and parenchymal staining in human and rat brain sections. Scale bars = 50 μm.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: GSK3β antibody immunostaining in cultured cells and tissue sections. (A,B) HEK293T cells stained with 12B2 ( A , green) and 15C2 ( B , green). ( C,D ) Undifferentiated SH-SY5Y cells stained with 12B2 ( C , green) and 15C2 ( D , green). (E,F) Rat primary cortical neurons (E18) stained with 12B2 ( E , green) and 15C2 ( F , green). In A-F , all cells were also stained with total GSK3α/β (red) and DAPI (blue in merged image). The pattern of staining with 12B2 and 15C2 was punctate staining throughout the cells, and 12B2 produced stronger signal than 15C2 in each cell type. Scale bars = 25 μm. (G,H) Brain sections in rat (left, retrosplenial cortex displayed) and human (right, temporal cortex) stained with 12B2 (G) or 15C2 (H) . In general, both antibodies produced clear somatodendritic and parenchymal staining in human and rat brain sections. Scale bars = 50 μm.

    Article Snippet: After sample incubation, the wells were rinsed and incubated in 50 μl/well GSK3β activity reaction buffer [100 μM ATP (V915A, Promega), 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2 and 0.1 mg/ml bovine serum albumin] for 1 h at 30°C.

    Techniques: Immunostaining, Cell Culture, Staining, Produced

    siRNA knockdown of GSK3α and GSK3β demonstrate specificity of the 15C2 antibody. (A) HEK293T cells were treated with control, GSK3α, GSK3β or GAPDH siRNAs and probed with 15C2 (red) and total GSK3β/α (green) antibodies. (B) Quantitation of 15C2 signal shows that GSK3α siRNA caused a loss of 84% for GSK3α and an increase in GSK3β (+22%) when compared to control cells. Quantitation of 15C2 shows that GSK3β siRNA caused a loss of 49% for GSK3β and an increase in GSK3α (+18%) when compared to control. (C) Quantitation of total GSK3α/β antibody signal shows that GSK3α siRNA caused a loss of 66% in GSK3α and an increase in GSK3β (+24%) when compared to controls. Quantitation of total GSK3α/β antibody signal shows that GSK3β siRNA caused a loss of 40% for the GSK3β and an increase in GSK3α (+9%) when compared to control cells. All immunoblotting data are normalized to GAPDH signal and expressed as percent of the control group to illustrate the siRNA-mediated changes in signal. (D) Immunocytofluorescence of HEK293T cells confirms the reduction in 15C2 detection of npS21 GSK3α or npS9 GSK3β when treated with GSK3α siRNA or GSK3β siRNA, respectively. Scale bars = 20 μm. Four independent experiments were performed. GAPDH siRNA quantitation is provided in Supplementary Figure S4 .

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: siRNA knockdown of GSK3α and GSK3β demonstrate specificity of the 15C2 antibody. (A) HEK293T cells were treated with control, GSK3α, GSK3β or GAPDH siRNAs and probed with 15C2 (red) and total GSK3β/α (green) antibodies. (B) Quantitation of 15C2 signal shows that GSK3α siRNA caused a loss of 84% for GSK3α and an increase in GSK3β (+22%) when compared to control cells. Quantitation of 15C2 shows that GSK3β siRNA caused a loss of 49% for GSK3β and an increase in GSK3α (+18%) when compared to control. (C) Quantitation of total GSK3α/β antibody signal shows that GSK3α siRNA caused a loss of 66% in GSK3α and an increase in GSK3β (+24%) when compared to controls. Quantitation of total GSK3α/β antibody signal shows that GSK3β siRNA caused a loss of 40% for the GSK3β and an increase in GSK3α (+9%) when compared to control cells. All immunoblotting data are normalized to GAPDH signal and expressed as percent of the control group to illustrate the siRNA-mediated changes in signal. (D) Immunocytofluorescence of HEK293T cells confirms the reduction in 15C2 detection of npS21 GSK3α or npS9 GSK3β when treated with GSK3α siRNA or GSK3β siRNA, respectively. Scale bars = 20 μm. Four independent experiments were performed. GAPDH siRNA quantitation is provided in Supplementary Figure S4 .

    Article Snippet: After sample incubation, the wells were rinsed and incubated in 50 μl/well GSK3β activity reaction buffer [100 μM ATP (V915A, Promega), 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2 and 0.1 mg/ml bovine serum albumin] for 1 h at 30°C.

    Techniques: Quantitation Assay

    12B2 and 15C2 are specific for nonphospho-Ser GSK3β/α peptides. Each antibody was screened in indirect ELISA titers against npS9 GSK3β, pS9 GSK3β, npS21 GSK3α and pS21 GSK3α peptides ( n = 3 independent experiments). (A) 12B2 showed strong reactivity for npS9 GSK3β compared to npS21 GSK3α peptides and did not react with pS9 or pS21 GSK3 peptides (EC 50 values: npS9 = 2.1 nM; pS9 = indeterminate (id); npS21 = 6.4 nM; pS21 = id). (B) To further confirm the specificity of 12B2, ELISAs were performed by coating wells with a wide range of peptide amounts (0 – 6.4 μg peptide/well). 12B2 showed strong reactivity with the npS GSK3 peptides (β > α), but did not react with pS GSK3 peptides. (C) 15C2 showed stronger reactivity for npS21 GSK3α compared to npS9 GSK3β and did not react with pS9 or pS21 GSK3 peptides (EC 50 values: npS9 = 2.4 nM; pS9 = id; npS21 = 277 pM; pS21 = id). (D) To further confirm the specificity of 15C2, ELISAs were performed by coating wells with a wide range of peptide amounts (0 – 6.4 μg peptide/well). 15C2 showed strong reactivity with the npS GSK3 peptides, but did not react with pS GSK peptides. It is noteworthy that synthetic peptides provide a homogeneous source of modified peptides, and thus, are ideal for challenging the specificity of the antibodies against nonphospho-Ser and phospho-Ser residues in GSK3.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: 12B2 and 15C2 are specific for nonphospho-Ser GSK3β/α peptides. Each antibody was screened in indirect ELISA titers against npS9 GSK3β, pS9 GSK3β, npS21 GSK3α and pS21 GSK3α peptides ( n = 3 independent experiments). (A) 12B2 showed strong reactivity for npS9 GSK3β compared to npS21 GSK3α peptides and did not react with pS9 or pS21 GSK3 peptides (EC 50 values: npS9 = 2.1 nM; pS9 = indeterminate (id); npS21 = 6.4 nM; pS21 = id). (B) To further confirm the specificity of 12B2, ELISAs were performed by coating wells with a wide range of peptide amounts (0 – 6.4 μg peptide/well). 12B2 showed strong reactivity with the npS GSK3 peptides (β > α), but did not react with pS GSK3 peptides. (C) 15C2 showed stronger reactivity for npS21 GSK3α compared to npS9 GSK3β and did not react with pS9 or pS21 GSK3 peptides (EC 50 values: npS9 = 2.4 nM; pS9 = id; npS21 = 277 pM; pS21 = id). (D) To further confirm the specificity of 15C2, ELISAs were performed by coating wells with a wide range of peptide amounts (0 – 6.4 μg peptide/well). 15C2 showed strong reactivity with the npS GSK3 peptides, but did not react with pS GSK peptides. It is noteworthy that synthetic peptides provide a homogeneous source of modified peptides, and thus, are ideal for challenging the specificity of the antibodies against nonphospho-Ser and phospho-Ser residues in GSK3.

    Article Snippet: After sample incubation, the wells were rinsed and incubated in 50 μl/well GSK3β activity reaction buffer [100 μM ATP (V915A, Promega), 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2 and 0.1 mg/ml bovine serum albumin] for 1 h at 30°C.

    Techniques: Indirect ELISA, Modification

    The Akt-protein phosphatase signaling pathway involved in regulating GSK3β phosphorylation. Active Akt (i.e., phosphorylated) inactivates GSK3β by phosphorylation at S9. Protein phosphatases can modulate GSK3β phosphorylation at S9 via two routes. (1) Protein phosphatases inactivate Akt by dephosphorylation, and (2) protein phosphatases activate GSK3β by directly dephosphorylating S9. Inhibition of Akt (with inhibitors such as AZD-5363) increases non-phosphorylated GSK3β by suppressing Akt-mediated phosphorylation of GSK3β. Inhibition of protein phosphatases (with inhibitors such as calyculin A) causes a decrease in non-phosphorylated GSK3β through the Akt pathway by increasing active Akt (the grayed portion of the Akt cycle). Protein phosphatase inhibition also leads to decreased non-phosphorylated GSK3β independent of Akt by directly dephosphorylating S9 in GSK3β. If an Akt inhibitor is applied followed by a protein phosphatase inhibitor the Akt-independent pathway can be evaluated.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: The Akt-protein phosphatase signaling pathway involved in regulating GSK3β phosphorylation. Active Akt (i.e., phosphorylated) inactivates GSK3β by phosphorylation at S9. Protein phosphatases can modulate GSK3β phosphorylation at S9 via two routes. (1) Protein phosphatases inactivate Akt by dephosphorylation, and (2) protein phosphatases activate GSK3β by directly dephosphorylating S9. Inhibition of Akt (with inhibitors such as AZD-5363) increases non-phosphorylated GSK3β by suppressing Akt-mediated phosphorylation of GSK3β. Inhibition of protein phosphatases (with inhibitors such as calyculin A) causes a decrease in non-phosphorylated GSK3β through the Akt pathway by increasing active Akt (the grayed portion of the Akt cycle). Protein phosphatase inhibition also leads to decreased non-phosphorylated GSK3β independent of Akt by directly dephosphorylating S9 in GSK3β. If an Akt inhibitor is applied followed by a protein phosphatase inhibitor the Akt-independent pathway can be evaluated.

    Article Snippet: After sample incubation, the wells were rinsed and incubated in 50 μl/well GSK3β activity reaction buffer [100 μM ATP (V915A, Promega), 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2 and 0.1 mg/ml bovine serum albumin] for 1 h at 30°C.

    Techniques: De-Phosphorylation Assay, Inhibition

    Detection of recombinant npS9 GSK3β with 12B2 and 15C2 antibodies is linear and correlates with kinase activity. (A) The level of GSK3β kinase activity with 30, 60, 120, 180, 240, and 300 ng of npS9 GSK3β (“active”) was measured using an in vitro GSK3β kinase activity assay and there was a linear increase in kinase activity with increasing amounts of GSK3β ( r 2 = 0.93). Three independent experiments were performed. (B) For western blotting, recombinant GSK3β was incubated with alkaline phosphatase to generate nonphosphoS9 GSK3β or incubated with Akt1 to generate phosphoS9 GSK3β, and then 0, 30, 60, 120, 180, 240, or 300 ng of npS9 GSK3β was mixed with 300, 240, 180, 120, 60, or 0 ng of pS9 GSK3β to bring the total protein content to 300 ng/lane. The blot was probed with 12B2 (red) and total GSK3α/β antibodies (green). (C) Quantitation of signal from 12B2 shows a linear increase in reactivity with increasing npS9 GSK3β amount ( r 2 = 0.92). (D) The same samples were probed with 15C2 (red) and total GSK3α/β antibodies (green). (E) Quantitation of signal from 15C2 shows a linear increase in reactivity with increasing npS9 GSK3β amount ( r 2 = 0.90). It is notable that both 12B2 and 15C2 signals also showed a direct correlation with GSK3β activity levels (12B2: r = 0.99, p = 0.0002; 15C2: r = 0.99, p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    doi: 10.3389/fnmol.2016.00123

    Figure Lengend Snippet: Detection of recombinant npS9 GSK3β with 12B2 and 15C2 antibodies is linear and correlates with kinase activity. (A) The level of GSK3β kinase activity with 30, 60, 120, 180, 240, and 300 ng of npS9 GSK3β (“active”) was measured using an in vitro GSK3β kinase activity assay and there was a linear increase in kinase activity with increasing amounts of GSK3β ( r 2 = 0.93). Three independent experiments were performed. (B) For western blotting, recombinant GSK3β was incubated with alkaline phosphatase to generate nonphosphoS9 GSK3β or incubated with Akt1 to generate phosphoS9 GSK3β, and then 0, 30, 60, 120, 180, 240, or 300 ng of npS9 GSK3β was mixed with 300, 240, 180, 120, 60, or 0 ng of pS9 GSK3β to bring the total protein content to 300 ng/lane. The blot was probed with 12B2 (red) and total GSK3α/β antibodies (green). (C) Quantitation of signal from 12B2 shows a linear increase in reactivity with increasing npS9 GSK3β amount ( r 2 = 0.92). (D) The same samples were probed with 15C2 (red) and total GSK3α/β antibodies (green). (E) Quantitation of signal from 15C2 shows a linear increase in reactivity with increasing npS9 GSK3β amount ( r 2 = 0.90). It is notable that both 12B2 and 15C2 signals also showed a direct correlation with GSK3β activity levels (12B2: r = 0.99, p = 0.0002; 15C2: r = 0.99, p

    Article Snippet: After sample incubation, the wells were rinsed and incubated in 50 μl/well GSK3β activity reaction buffer [100 μM ATP (V915A, Promega), 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2 and 0.1 mg/ml bovine serum albumin] for 1 h at 30°C.

    Techniques: Recombinant, Activity Assay, In Vitro, Kinase Assay, Western Blot, Incubation, Quantitation Assay

    Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: MTT Assay, Expressing, Western Blot, Activity Assay, Double Staining, FACS, Fluorescence, Staining

    Treatment with a ROS scavenger suppresses doxorubicin (DOX)-induced cytosolic adenosine triphosphate (cATP) production, DNA damage, mitochondrial hyper-activation, and necrosis. HK-2 cells were treated with a ROS scavenger (N-acetyl cysteine, NAC, 5 mM), DOX (1 μM), or both DOX and NAC for 72 h. ( A , B ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, staining, respectively, and signals were detected by FACS analysis. ( C ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), and H2AX were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. The gels were electrophoresed under the same experimental conditions. ( D , E ) Mitochondrial respiration and cATP production were measured using MitoTracker Red CMXRos staining and ATP-based CellTiter-Glo Luminescent Cell Viability Assays, and signals were detected using FACS analysis or analysis on a microplate reader, respectively. ( F ) Morphological changes were measured using phase-contrast microscopy (original magnification, 100×). ( G ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected using FACS analysis. The histogram shows the percentage of cells in each group, as the average of the results of three independent experiments (* P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Treatment with a ROS scavenger suppresses doxorubicin (DOX)-induced cytosolic adenosine triphosphate (cATP) production, DNA damage, mitochondrial hyper-activation, and necrosis. HK-2 cells were treated with a ROS scavenger (N-acetyl cysteine, NAC, 5 mM), DOX (1 μM), or both DOX and NAC for 72 h. ( A , B ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, staining, respectively, and signals were detected by FACS analysis. ( C ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), and H2AX were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. The gels were electrophoresed under the same experimental conditions. ( D , E ) Mitochondrial respiration and cATP production were measured using MitoTracker Red CMXRos staining and ATP-based CellTiter-Glo Luminescent Cell Viability Assays, and signals were detected using FACS analysis or analysis on a microplate reader, respectively. ( F ) Morphological changes were measured using phase-contrast microscopy (original magnification, 100×). ( G ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected using FACS analysis. The histogram shows the percentage of cells in each group, as the average of the results of three independent experiments (* P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: Activation Assay, Staining, FACS, Expressing, Western Blot, Microscopy, Double Staining

    Inhibition of PARP1 suppresses doxorubicin (DOX)-induced DNA damage, mitochondrial hyper-activation, and necrosis. ( A–J ) HK-2 cells were treated with a pharmacological PARP1 inhibitor (PJ-34, 10 μM), DOX (1 μM), or both PJ-34 and DOX for 72 h. (A) Expressions of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), H2AX (15 kDa), 53BP1 (350 kDa), p-ATM ser1981 (350 kDa), ATM (350 kDa), p-CHK2 Thr68 (62 kDa), and CHK2 (62 kDa) proteins were measured by western blot in whole cell extracts. ( B ) Levels of p-ATM ser1981 and γ-H2AX ser139 in the nucleus were measured using immunofluorescence staining and Cellomics ArrayScan HCS Reader. ( C ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected by FACS. ( D ) Mitochondrial respiration levels were measured using MitoTracker Red CMXRos and signals were detected using FACS. ( E ) Genomic DNA was analyzed for mtDNA copy numbers using ND1- , ND4- , and GAPDH- specific primers and real-time PCR. ND1 and ND4 copy numbers were normalized to GAPDH copy numbers. ( F ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. ( G , H ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, respectively, and signals were detected by FACS. (I and J) Cytosolic NO and secreted NO levels were measured using DAF-FM and an NO detection kit. ( K , N ) HK-2 cells were transfected with SC-siRNA (5 nM) or PARP1 -siRNA (5 nM) and treated with DOX for 72 h. ( K ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assay. ( L ) Cytosolic ROS levels were measured using DCF-DA and were detected using FACS. ( M ) Cell viability was measured using MTT assay, and signals were detected by a microplate reader. ( N ) Necrotic cell death was measured using PI staining, and signals were detected by FACS. (* P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Inhibition of PARP1 suppresses doxorubicin (DOX)-induced DNA damage, mitochondrial hyper-activation, and necrosis. ( A–J ) HK-2 cells were treated with a pharmacological PARP1 inhibitor (PJ-34, 10 μM), DOX (1 μM), or both PJ-34 and DOX for 72 h. (A) Expressions of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), H2AX (15 kDa), 53BP1 (350 kDa), p-ATM ser1981 (350 kDa), ATM (350 kDa), p-CHK2 Thr68 (62 kDa), and CHK2 (62 kDa) proteins were measured by western blot in whole cell extracts. ( B ) Levels of p-ATM ser1981 and γ-H2AX ser139 in the nucleus were measured using immunofluorescence staining and Cellomics ArrayScan HCS Reader. ( C ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected by FACS. ( D ) Mitochondrial respiration levels were measured using MitoTracker Red CMXRos and signals were detected using FACS. ( E ) Genomic DNA was analyzed for mtDNA copy numbers using ND1- , ND4- , and GAPDH- specific primers and real-time PCR. ND1 and ND4 copy numbers were normalized to GAPDH copy numbers. ( F ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. ( G , H ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, respectively, and signals were detected by FACS. (I and J) Cytosolic NO and secreted NO levels were measured using DAF-FM and an NO detection kit. ( K , N ) HK-2 cells were transfected with SC-siRNA (5 nM) or PARP1 -siRNA (5 nM) and treated with DOX for 72 h. ( K ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assay. ( L ) Cytosolic ROS levels were measured using DCF-DA and were detected using FACS. ( M ) Cell viability was measured using MTT assay, and signals were detected by a microplate reader. ( N ) Necrotic cell death was measured using PI staining, and signals were detected by FACS. (* P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: Inhibition, Activation Assay, Western Blot, Immunofluorescence, Staining, Double Staining, FACS, Real-time Polymerase Chain Reaction, Transfection, Cell Viability Assay, MTT Assay

    Adenosine triphosphate (ATP) treatment induces DNA damage and necrosis at the early stage and apoptosis at the late stage, but does not alter cytosolic reactive oxygen species (cROS) and mitochondrial reactive oxygen species (mROS) levels. HK-2 cells were treated with ATP (1 mM) for 24, 48, and 72 h. ( A ) Cell viability was measured using MTT assays. HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Cytosolic ATP was measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. Signals were detected using a microplate reader. ( B ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa and 55 kDa), γ-H2AX ser139 (15 kDa), and H2AX (15 kDa) proteins were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Caspase 3/7 activity was measured by Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. Representative images are shown. ( D , E ) cROS, mROS, and cytosolic NO levels were measured using DCF-DA, MitoSOX, and DAF-FM staining, respectively, and signals were detected by FACS analysis. ( F ) Cell death, including necrosis (PI-positive cells) and apoptosis (Annexin V-positive cells), was measured using Annexin V or PI staining. Signals were detected by FACS analysis. The histogram shows the percentage of PI- and Annexin V-positive cells, as the average of the results of three independent experiments (*P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Adenosine triphosphate (ATP) treatment induces DNA damage and necrosis at the early stage and apoptosis at the late stage, but does not alter cytosolic reactive oxygen species (cROS) and mitochondrial reactive oxygen species (mROS) levels. HK-2 cells were treated with ATP (1 mM) for 24, 48, and 72 h. ( A ) Cell viability was measured using MTT assays. HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Cytosolic ATP was measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. Signals were detected using a microplate reader. ( B ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa and 55 kDa), γ-H2AX ser139 (15 kDa), and H2AX (15 kDa) proteins were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Caspase 3/7 activity was measured by Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. Representative images are shown. ( D , E ) cROS, mROS, and cytosolic NO levels were measured using DCF-DA, MitoSOX, and DAF-FM staining, respectively, and signals were detected by FACS analysis. ( F ) Cell death, including necrosis (PI-positive cells) and apoptosis (Annexin V-positive cells), was measured using Annexin V or PI staining. Signals were detected by FACS analysis. The histogram shows the percentage of PI- and Annexin V-positive cells, as the average of the results of three independent experiments (*P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: MTT Assay, Expressing, Western Blot, Activity Assay, Staining, FACS

    Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase PCR, using primers from 5 BLV genome regions, was used to amplify products from DNA extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.

    Journal: Emerging Infectious Diseases

    Article Title: Bovine Leukemia Virus DNA in Human Breast Tissue

    doi: 10.3201/eid2005.131298

    Figure Lengend Snippet: Amplification of bovine leukemia virus (BLV) genome regions in human breast tissue specimens. Nested liquid-phase PCR, using primers from 5 BLV genome regions, was used to amplify products from DNA extracted from breast tissues of 6 human donors. PCR products for each tissue were loaded into 1 well and separated by agarose gel (3.5%) electrophoresis on the basis of size differences: long terminal repeat, 290 bp; group-specific antigen, 272 bp; envelope, 230 bp; trans-activating gene of the X region, 206 bp; polymerase, 157 bp. The section below the white line shows the glyceraldehyde 3-phosphate dehydrogenase amplification of each sample as an indicator of DNA quality. Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA); lane 2, fetal lamb kidney cell line, positive control; lane 3, no-template-DNA negative control (water substituted for DNA template); lane 4, human sample 143; lane 5, human sample 236; lane 6, human sample 010; lane 7, human sample 20874; lane 8, human sample 23803; lane 9, human sample 0253.

    Article Snippet: For nested L-PCR, extracted DNA (0.85 µg) was added to 50 μL of PCR mix (2.0 mmol/L MgCl2 , 0.2 mmol/L dNTPs, 0.025 U/µL Taq polymerase [all from Promega, Madison, WI, USA], and 0.2 μmol/L outer primers for each BLV gene [ ]) in Hot Start Micro 50 tubes (MolecularBio Products, San Diego, CA, USA).

    Techniques: Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Molecular Weight, Marker, Positive Control, Negative Control

    Test results showing lack of cross - reactivity of bovine leukemia virus (BLV)–specific primers with representatives of all mammalian and avian retrovirus subfamilies and human exogenous and endogenous viruses previously identified in human breast tissue. Nested liquid-phase PCR used primers from 5 BLV genome regions with template DNA from the viruses in lanes 4–10 and 12–21. PCR products for each virus, loaded into 1 well, were separated by agarose gel (1.5%) electrophoresis on the basis of size differences. Amplicons were generated only for known BLV-positive cell lines (FLK and Bat 2 Cl 6 ). Samples in lanes 13–21 were run simultaneously in the same gel in wells below samples in lanes 4–12. The section below the white line shows glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification of each sample to indicate DNA quality. Human GAPDH primers were used for human, rhesus monkey, baboon, and bat cell lines (amplicon = 237 bp); murine GAPDH for mouse and rat cell lines (796 bp); and bovine GAPDH for bovine, ovine, and feline cell lines (857 bp). Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA), lane 2, fetal lamb kidney cell line, positive control; lane 3, water substituted for DNA template, negative control; lane 4, Rous sarcoma virus; lane 5, murine sarcoma virus; lane 6, mouse mammary tumor virus; lane 7, Mason-Pfizer monkey virus; lane 8, murine leukemia virus; lane 9, feline leukemia virus; lane 10, BLV Bat 2 Cl 6 ; lane 11, Tb 1 Lu (known BLV-negative cell line), negative control; lane 12, simian T-cell leukemia virus; lane 13, human T-cell leukemia virus 1; lane 14, human T-cell leukemia virus 2; lane 15, HIV-1; lane 16, HIV-2; lane 17, human papillomavirus 16; lane 18, human papillomavirus 18; lane 19, Epstein-Barr virus; lane 20, human endogenous retrovirus K; lane 21, env of human endogenous retrovirus K.

    Journal: Emerging Infectious Diseases

    Article Title: Bovine Leukemia Virus DNA in Human Breast Tissue

    doi: 10.3201/eid2005.131298

    Figure Lengend Snippet: Test results showing lack of cross - reactivity of bovine leukemia virus (BLV)–specific primers with representatives of all mammalian and avian retrovirus subfamilies and human exogenous and endogenous viruses previously identified in human breast tissue. Nested liquid-phase PCR used primers from 5 BLV genome regions with template DNA from the viruses in lanes 4–10 and 12–21. PCR products for each virus, loaded into 1 well, were separated by agarose gel (1.5%) electrophoresis on the basis of size differences. Amplicons were generated only for known BLV-positive cell lines (FLK and Bat 2 Cl 6 ). Samples in lanes 13–21 were run simultaneously in the same gel in wells below samples in lanes 4–12. The section below the white line shows glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification of each sample to indicate DNA quality. Human GAPDH primers were used for human, rhesus monkey, baboon, and bat cell lines (amplicon = 237 bp); murine GAPDH for mouse and rat cell lines (796 bp); and bovine GAPDH for bovine, ovine, and feline cell lines (857 bp). Lane 1, molecular weight marker (HyperLadder IV; Bioline, Taunton, MA, USA), lane 2, fetal lamb kidney cell line, positive control; lane 3, water substituted for DNA template, negative control; lane 4, Rous sarcoma virus; lane 5, murine sarcoma virus; lane 6, mouse mammary tumor virus; lane 7, Mason-Pfizer monkey virus; lane 8, murine leukemia virus; lane 9, feline leukemia virus; lane 10, BLV Bat 2 Cl 6 ; lane 11, Tb 1 Lu (known BLV-negative cell line), negative control; lane 12, simian T-cell leukemia virus; lane 13, human T-cell leukemia virus 1; lane 14, human T-cell leukemia virus 2; lane 15, HIV-1; lane 16, HIV-2; lane 17, human papillomavirus 16; lane 18, human papillomavirus 18; lane 19, Epstein-Barr virus; lane 20, human endogenous retrovirus K; lane 21, env of human endogenous retrovirus K.

    Article Snippet: For nested L-PCR, extracted DNA (0.85 µg) was added to 50 μL of PCR mix (2.0 mmol/L MgCl2 , 0.2 mmol/L dNTPs, 0.025 U/µL Taq polymerase [all from Promega, Madison, WI, USA], and 0.2 μmol/L outer primers for each BLV gene [ ]) in Hot Start Micro 50 tubes (MolecularBio Products, San Diego, CA, USA).

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Electrophoresis, Generated, Amplification, Molecular Weight, Marker, Positive Control, Negative Control

    Activation of MEK/ERK signalling by hypoactive BRAF mutants is RAS-dependent a , V5-tagged wild-type (WT) or mutant BRAF kinases were expressed in 293H cells that stably express NRAS(Q61K). These BRAF protein kinases were isolated with anti-V5 agarose. The in vitro . b . c , In vitro kinase activity of the indicated BRAF proteins which were isolated from 293H cells that stably express NRAS(Q61K) was assessed as described in a . d . e , NIH3T3 cells expressing the indicated BRAF proteins were stimulated with 100 ng ml −1 . f were cultured in doxycycline (30 ng ml −1 . g . h , The frequency of coexistent RAS or NF1 alterations in human BRAF mutant melanomas compared to that in NSCLC and colorectal cancers.. The calculation was based on the sample sets as shown in . The P values were calculated by using a paired t -test. N.S., not significant. i , The phosphorylation of multiple RTKs in the indicated cell lines was assayed using the Human Phospho-RTK Array Kit. Phosphorylated RTKs are highlighted with boxes in different colours. j . k , Cells were cultured and exposed to cetuximab at concentrations of 0, 0.3, 1, 3, 10, 30, 100 and 300 nM for 3 days. The effects of drug on cell growth were quantified using the ATP-Glo assay. Graphs were generated using Prism 6 (mean ±s.d. are represented by the dots and error bars, n =8). l . m , The effects of cetuximab or trametinib on the growth of the cells described in l was determined by ATP-Glo assay after 3 days treatment. Graphs were generated using Prism 6 (mean ± s.d., n = 8). n . o , Growth inhibition of the cells described in n after three days exposure to varying doses of cetuximab on day 3 was determined by ATP-Glo assay. Graphs were generated using Prism 6 (mean ± s.d., n =8) Fig. 1d

    Journal:

    Article Title: Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS

    doi: 10.1038/nature23291

    Figure Lengend Snippet: Activation of MEK/ERK signalling by hypoactive BRAF mutants is RAS-dependent a , V5-tagged wild-type (WT) or mutant BRAF kinases were expressed in 293H cells that stably express NRAS(Q61K). These BRAF protein kinases were isolated with anti-V5 agarose. The in vitro . b . c , In vitro kinase activity of the indicated BRAF proteins which were isolated from 293H cells that stably express NRAS(Q61K) was assessed as described in a . d . e , NIH3T3 cells expressing the indicated BRAF proteins were stimulated with 100 ng ml −1 . f were cultured in doxycycline (30 ng ml −1 . g . h , The frequency of coexistent RAS or NF1 alterations in human BRAF mutant melanomas compared to that in NSCLC and colorectal cancers.. The calculation was based on the sample sets as shown in . The P values were calculated by using a paired t -test. N.S., not significant. i , The phosphorylation of multiple RTKs in the indicated cell lines was assayed using the Human Phospho-RTK Array Kit. Phosphorylated RTKs are highlighted with boxes in different colours. j . k , Cells were cultured and exposed to cetuximab at concentrations of 0, 0.3, 1, 3, 10, 30, 100 and 300 nM for 3 days. The effects of drug on cell growth were quantified using the ATP-Glo assay. Graphs were generated using Prism 6 (mean ±s.d. are represented by the dots and error bars, n =8). l . m , The effects of cetuximab or trametinib on the growth of the cells described in l was determined by ATP-Glo assay after 3 days treatment. Graphs were generated using Prism 6 (mean ± s.d., n = 8). n . o , Growth inhibition of the cells described in n after three days exposure to varying doses of cetuximab on day 3 was determined by ATP-Glo assay. Graphs were generated using Prism 6 (mean ± s.d., n =8) Fig. 1d

    Article Snippet: Cell growth was quantified using the ATP-Glo assay (Promega, G7572) every 24 h. For each condition, 8 replicates at each concentration were measured.

    Techniques: Activation Assay, Mutagenesis, Stable Transfection, Isolation, In Vitro, Activity Assay, Expressing, Cell Culture, Glo Assay, Generated, Inhibition

    ERK signalling in hypoactive BRAF-mutant cells is sensitive to trametinib a . b , The cell lines as indicated in a were treated with different concentrations of trametinib for 3 days. Cell viability was determined by ATP-Glo assay. Dose-dependent inhibition curves were generated using Prism 6 (mean ± s.d., n =8).

    Journal:

    Article Title: Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS

    doi: 10.1038/nature23291

    Figure Lengend Snippet: ERK signalling in hypoactive BRAF-mutant cells is sensitive to trametinib a . b , The cell lines as indicated in a were treated with different concentrations of trametinib for 3 days. Cell viability was determined by ATP-Glo assay. Dose-dependent inhibition curves were generated using Prism 6 (mean ± s.d., n =8).

    Article Snippet: Cell growth was quantified using the ATP-Glo assay (Promega, G7572) every 24 h. For each condition, 8 replicates at each concentration were measured.

    Techniques: Mutagenesis, Glo Assay, Inhibition, Generated

    In hypoactive BRAF-mutant tumours with wild-type RAS/NF1 ERK signalling is sensitive to upstream inhibition of RAS a . b , RTK phosphorylation profile of the cells isolated from the ovarian metastatisis derived PDX (BRCC-OVA) was assessed with Human Phospho-RTK arrays. The elevated p-EGFR and p-MET bands are within the red and green rectangles, respectively. c . d , e . f , The growth inhibition of BRCC-OVA cells with indicated drugs at 0, 1, 3, 10, 30, 100, 300 and 1,000 nM of trametinib or 0, 10, 30, 100, 300, 1,000, 3,000 and 10,000 nM of INC280 or combination of increasing dose of trametinib with 100 nM INC280 was determined by ATP-Glo assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ±s.d., n = 8). Fig. 4b

    Journal:

    Article Title: Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS

    doi: 10.1038/nature23291

    Figure Lengend Snippet: In hypoactive BRAF-mutant tumours with wild-type RAS/NF1 ERK signalling is sensitive to upstream inhibition of RAS a . b , RTK phosphorylation profile of the cells isolated from the ovarian metastatisis derived PDX (BRCC-OVA) was assessed with Human Phospho-RTK arrays. The elevated p-EGFR and p-MET bands are within the red and green rectangles, respectively. c . d , e . f , The growth inhibition of BRCC-OVA cells with indicated drugs at 0, 1, 3, 10, 30, 100, 300 and 1,000 nM of trametinib or 0, 10, 30, 100, 300, 1,000, 3,000 and 10,000 nM of INC280 or combination of increasing dose of trametinib with 100 nM INC280 was determined by ATP-Glo assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ±s.d., n = 8). Fig. 4b

    Article Snippet: Cell growth was quantified using the ATP-Glo assay (Promega, G7572) every 24 h. For each condition, 8 replicates at each concentration were measured.

    Techniques: Mutagenesis, Inhibition, Isolation, Derivative Assay, Glo Assay, Generated