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    Structured Review

    Millipore β mercaptoethanol
    Sensitivity of alanine block CydX mutants to <t>β-mercaptoethanol.</t> A) Amino acid sequences of alanine block mutant CydX mutants. B) Zones of growth inhibition for wild-type, Δ cydX , and the three alanine block cydX mutants. The “Block 1” mutant contains alanines from positions 2 to 11, the “Block 2” contains alanines from positions 12–21, and the “Block 3” contains alanines from positions 22 to 31. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    β Mercaptoethanol, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2080 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level"

    Article Title: Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198699

    Sensitivity of alanine block CydX mutants to β-mercaptoethanol. A) Amino acid sequences of alanine block mutant CydX mutants. B) Zones of growth inhibition for wild-type, Δ cydX , and the three alanine block cydX mutants. The “Block 1” mutant contains alanines from positions 2 to 11, the “Block 2” contains alanines from positions 12–21, and the “Block 3” contains alanines from positions 22 to 31. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of alanine block CydX mutants to β-mercaptoethanol. A) Amino acid sequences of alanine block mutant CydX mutants. B) Zones of growth inhibition for wild-type, Δ cydX , and the three alanine block cydX mutants. The “Block 1” mutant contains alanines from positions 2 to 11, the “Block 2” contains alanines from positions 12–21, and the “Block 3” contains alanines from positions 22 to 31. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Blocking Assay, Mutagenesis, Inhibition

    Sensitivity of single amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for wild-type, Δ cydX and the cydX +Kan template strain used to make the mutants. B) Zones of growth inhibition for each single mutant strain. The E . coli CydX protein sequence is inset. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of single amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for wild-type, Δ cydX and the cydX +Kan template strain used to make the mutants. B) Zones of growth inhibition for each single mutant strain. The E . coli CydX protein sequence is inset. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Inhibition, Mutagenesis, Sequencing

    Sensitivity of double and triple amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for double and triple mutants. B) Growth of select double and triple cydX mutants in liquid culture containing β-mercaptoethanol. Samples are as follows: wild-type (filled circles), Δ cydX mutant (open circles), cydX+KAN (filled triangle), W2A/G9A mutant (open triangle), Y3A/G9A mutant (filled square), W6A/G9A mutant (open square), W2A/W6A/G9A mutant (closed diamond), Y3A/W6A/G9A mutant (open diamond), Y3A/G9A/L10G mutant (closed triangle). Strains with growth curves similar to wild-type are delineated by a dark line, strains with a growth phenotype similar to the deletion mutant are delineated with a dotted line, and strains with an intermediate growth phenotype are delineated by a grey line. Liquid culture experiments were conducted in Luria Broth containing 20mM β-mercaptoethanol. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of double and triple amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for double and triple mutants. B) Growth of select double and triple cydX mutants in liquid culture containing β-mercaptoethanol. Samples are as follows: wild-type (filled circles), Δ cydX mutant (open circles), cydX+KAN (filled triangle), W2A/G9A mutant (open triangle), Y3A/G9A mutant (filled square), W6A/G9A mutant (open square), W2A/W6A/G9A mutant (closed diamond), Y3A/W6A/G9A mutant (open diamond), Y3A/G9A/L10G mutant (closed triangle). Strains with growth curves similar to wild-type are delineated by a dark line, strains with a growth phenotype similar to the deletion mutant are delineated with a dotted line, and strains with an intermediate growth phenotype are delineated by a grey line. Liquid culture experiments were conducted in Luria Broth containing 20mM β-mercaptoethanol. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Inhibition, Mutagenesis

    2) Product Images from "Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level"

    Article Title: Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198699

    Sensitivity of alanine block CydX mutants to β-mercaptoethanol. A) Amino acid sequences of alanine block mutant CydX mutants. B) Zones of growth inhibition for wild-type, Δ cydX , and the three alanine block cydX mutants. The “Block 1” mutant contains alanines from positions 2 to 11, the “Block 2” contains alanines from positions 12–21, and the “Block 3” contains alanines from positions 22 to 31. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of alanine block CydX mutants to β-mercaptoethanol. A) Amino acid sequences of alanine block mutant CydX mutants. B) Zones of growth inhibition for wild-type, Δ cydX , and the three alanine block cydX mutants. The “Block 1” mutant contains alanines from positions 2 to 11, the “Block 2” contains alanines from positions 12–21, and the “Block 3” contains alanines from positions 22 to 31. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Blocking Assay, Mutagenesis, Inhibition

    Sensitivity of single amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for wild-type, Δ cydX and the cydX +Kan template strain used to make the mutants. B) Zones of growth inhibition for each single mutant strain. The E . coli CydX protein sequence is inset. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of single amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for wild-type, Δ cydX and the cydX +Kan template strain used to make the mutants. B) Zones of growth inhibition for each single mutant strain. The E . coli CydX protein sequence is inset. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Inhibition, Mutagenesis, Sequencing

    Sensitivity of double and triple amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for double and triple mutants. B) Growth of select double and triple cydX mutants in liquid culture containing β-mercaptoethanol. Samples are as follows: wild-type (filled circles), Δ cydX mutant (open circles), cydX+KAN (filled triangle), W2A/G9A mutant (open triangle), Y3A/G9A mutant (filled square), W6A/G9A mutant (open square), W2A/W6A/G9A mutant (closed diamond), Y3A/W6A/G9A mutant (open diamond), Y3A/G9A/L10G mutant (closed triangle). Strains with growth curves similar to wild-type are delineated by a dark line, strains with a growth phenotype similar to the deletion mutant are delineated with a dotted line, and strains with an intermediate growth phenotype are delineated by a grey line. Liquid culture experiments were conducted in Luria Broth containing 20mM β-mercaptoethanol. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.
    Figure Legend Snippet: Sensitivity of double and triple amino acid CydX mutants to β-mercaptoethanol. A) Zones of growth inhibition for double and triple mutants. B) Growth of select double and triple cydX mutants in liquid culture containing β-mercaptoethanol. Samples are as follows: wild-type (filled circles), Δ cydX mutant (open circles), cydX+KAN (filled triangle), W2A/G9A mutant (open triangle), Y3A/G9A mutant (filled square), W6A/G9A mutant (open square), W2A/W6A/G9A mutant (closed diamond), Y3A/W6A/G9A mutant (open diamond), Y3A/G9A/L10G mutant (closed triangle). Strains with growth curves similar to wild-type are delineated by a dark line, strains with a growth phenotype similar to the deletion mutant are delineated with a dotted line, and strains with an intermediate growth phenotype are delineated by a grey line. Liquid culture experiments were conducted in Luria Broth containing 20mM β-mercaptoethanol. All experiments were performed in at least triplicate, and the standard error of each experiment is shown.

    Techniques Used: Inhibition, Mutagenesis

    3) Product Images from "Conservation analysis of the CydX protein yields insights into small protein identification and evolution"

    Article Title: Conservation analysis of the CydX protein yields insights into small protein identification and evolution

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-946

    Confirmation of functionality of CydX homologues. (A) Alignment of protein sequences of CydX homologues from Escherichia coli and other bacteria species. The small protein from Burkholderia sp. 383 (“Burkholderia383”) is not thought to be a homologue and was included as a negative control for the assay. Based on its significant sequence divergence was included in a separate alignment. (B) Alignment of the E. coli CydX protein with the CydZ protein from Klebsiella pneumoniae . (C) Assay of complementation of the Δ cydX β-mercaptoethanol sensitivity phenotype by expression of potential CydX homologues, a false positive from the tblastn search ( Burkholderia sp. 383 ), and an unrelated small protein (CydZ) from a different bacterial species. Sensitivity was measured using zones of inhibition, and the diameter of the zone after addition of 10 μL of 12 M β-mercaptoethanol to a plate of bacteria is shown. Species are as follows: Escherichia coli (“Escherichia”), Pectobacterium atrosepticus (“Pectobacterium”), Burkholderia xenovorans (“Burkholderia”), Actinobacillus pleuropneumoniae (“Actinobacillus”), Burkholderia sp. 383 (“Burkholderia sp. 383”), Klebsiella pneumoniae (“Klebsiella”), Cellvibrio japonicus Ueda107 (“Cellvibrio”), Methylibium petroleiphilum PM1 (“Methylibium”), Haemophilus influenzae 10810 (“Haemophilus”), and Francisella philomiragia subsp. Philomiragia ATCC 25017 (“Francisella”). Alignments were generated using the program MUSCLE [ 57 ]. Amino acids are colored based on their properties at physiological conditions as follows: red amino acids are hydrophobic, green residues are hydrophilic, purple residues are positively-charged and blue residues are negatively-charged. ‘*’ indicates that the residues are identical in all sequences and ‘:’ and ‘.’, respectively, indicated conserved and semi-conserved substitutions as defined by MUSCLE.
    Figure Legend Snippet: Confirmation of functionality of CydX homologues. (A) Alignment of protein sequences of CydX homologues from Escherichia coli and other bacteria species. The small protein from Burkholderia sp. 383 (“Burkholderia383”) is not thought to be a homologue and was included as a negative control for the assay. Based on its significant sequence divergence was included in a separate alignment. (B) Alignment of the E. coli CydX protein with the CydZ protein from Klebsiella pneumoniae . (C) Assay of complementation of the Δ cydX β-mercaptoethanol sensitivity phenotype by expression of potential CydX homologues, a false positive from the tblastn search ( Burkholderia sp. 383 ), and an unrelated small protein (CydZ) from a different bacterial species. Sensitivity was measured using zones of inhibition, and the diameter of the zone after addition of 10 μL of 12 M β-mercaptoethanol to a plate of bacteria is shown. Species are as follows: Escherichia coli (“Escherichia”), Pectobacterium atrosepticus (“Pectobacterium”), Burkholderia xenovorans (“Burkholderia”), Actinobacillus pleuropneumoniae (“Actinobacillus”), Burkholderia sp. 383 (“Burkholderia sp. 383”), Klebsiella pneumoniae (“Klebsiella”), Cellvibrio japonicus Ueda107 (“Cellvibrio”), Methylibium petroleiphilum PM1 (“Methylibium”), Haemophilus influenzae 10810 (“Haemophilus”), and Francisella philomiragia subsp. Philomiragia ATCC 25017 (“Francisella”). Alignments were generated using the program MUSCLE [ 57 ]. Amino acids are colored based on their properties at physiological conditions as follows: red amino acids are hydrophobic, green residues are hydrophilic, purple residues are positively-charged and blue residues are negatively-charged. ‘*’ indicates that the residues are identical in all sequences and ‘:’ and ‘.’, respectively, indicated conserved and semi-conserved substitutions as defined by MUSCLE.

    Techniques Used: Negative Control, Sequencing, Expressing, Inhibition, Generated

    Testing the functional importance of the CydX C-terminal amino acids. (A) Alignment of the E. coli CydX protein sequence along with six mutant sequences containing mutated C-terminal amino acid sequences. (B) Assay of CydX function was conducted using a zone assay testing the sensitivity to β-mercaptoethanol. Sensitivity was measured using zones of inhibition, and the diameter of the zone after addition of 10 μL of 12 M β-mercaptoethanol to a plate of bacteria is shown. The average and standard deviation of zone sizes was calculated from at least three replicate plates. Alignments were generated using the program MUSCLE [ 57 ].
    Figure Legend Snippet: Testing the functional importance of the CydX C-terminal amino acids. (A) Alignment of the E. coli CydX protein sequence along with six mutant sequences containing mutated C-terminal amino acid sequences. (B) Assay of CydX function was conducted using a zone assay testing the sensitivity to β-mercaptoethanol. Sensitivity was measured using zones of inhibition, and the diameter of the zone after addition of 10 μL of 12 M β-mercaptoethanol to a plate of bacteria is shown. The average and standard deviation of zone sizes was calculated from at least three replicate plates. Alignments were generated using the program MUSCLE [ 57 ].

    Techniques Used: Functional Assay, Sequencing, Mutagenesis, Inhibition, Standard Deviation, Generated

    4) Product Images from "Investigating the mincing method for isolation of adipose-derived stem cells from pregnant women fat"

    Article Title: Investigating the mincing method for isolation of adipose-derived stem cells from pregnant women fat

    Journal: Cytotechnology

    doi: 10.1007/s10616-017-0162-8

    The morphology and RT-PCR analyses of the expression of insulin producing cells-related genes in P-ADSCs during the induced differentiation process. The step 1 decrease the FBS concentration from 10% to 2% and supplemented wtih NEAA and β-mercaptoethanol, then step 2 supplement with activin A, nicotinamide and bFGF, EGF, finally step 3 activin A, nicotinamide, and exendin 4 was supplied. a The changes in cell morphology could be observed from day 2 to day 16 after induction. b Expression analysis of islet markers using RT-PCR in pADSC after β-cell differentiation with indicated protocols. Scale bar 100 μm for micrograph
    Figure Legend Snippet: The morphology and RT-PCR analyses of the expression of insulin producing cells-related genes in P-ADSCs during the induced differentiation process. The step 1 decrease the FBS concentration from 10% to 2% and supplemented wtih NEAA and β-mercaptoethanol, then step 2 supplement with activin A, nicotinamide and bFGF, EGF, finally step 3 activin A, nicotinamide, and exendin 4 was supplied. a The changes in cell morphology could be observed from day 2 to day 16 after induction. b Expression analysis of islet markers using RT-PCR in pADSC after β-cell differentiation with indicated protocols. Scale bar 100 μm for micrograph

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Concentration Assay, Cell Differentiation

    5) Product Images from "Ethanol Cellular Defense Induce Unfolded Protein Response in Yeast"

    Article Title: Ethanol Cellular Defense Induce Unfolded Protein Response in Yeast

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.00189

    Tolerance to the protein denaturing agent BME in CECT10094 and PE35M strains. (A) Drop test analysis in GPY media and in presence of 5 or 15 mM BME. The plates were grown for 6 days at 28°C. (B) Estimation of the minimum inhibitory concentration (empty arrows) and the non-inhibitory concentration (black arrows) of the CECT10094 and Temohaya-MI26 strains to protein denaturing agent β-mercaptoethanol. Curve fitting was achieved with a modified Gompertz function for decay.
    Figure Legend Snippet: Tolerance to the protein denaturing agent BME in CECT10094 and PE35M strains. (A) Drop test analysis in GPY media and in presence of 5 or 15 mM BME. The plates were grown for 6 days at 28°C. (B) Estimation of the minimum inhibitory concentration (empty arrows) and the non-inhibitory concentration (black arrows) of the CECT10094 and Temohaya-MI26 strains to protein denaturing agent β-mercaptoethanol. Curve fitting was achieved with a modified Gompertz function for decay.

    Techniques Used: Concentration Assay, Modification

    6) Product Images from "p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons"

    Article Title: p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0690-04.2004

    p35/Cdk5 and RasGRF2 mediate cellular Rac activity and ERK1/2 activity. A , CHO cells were transfected with EV-GTP (lane 1), RasGRF2 (lane 2), RasGRF2+p35+Cdk5 (lane 3), RasGRF2+Cdk5 (lane4), and EV+GTP (lane5). CHO cell lysates were incubated with GST-PAK1 and GST beads for 1 hr. Beads with bound active Rac were washed three times in lysis–binding–wash buffer and eluted by addition of 2× SDS-sample buffer/β-mercaptoethanol and boiling for 5 min. Eluted, active Rac samples were resolved by SDS-PAGE, transferred onto nitrocellulose, and immunodetected using the monoclonal anti-Rac1 Ab provided in the kit at 1:1000 dilution. p35/Cdk5 reduced RasGRF2-mediated active Rac levels. EV lysate incubated with GTP before the pull-downs exhibited the highest active Rac levels (EV+GTP). Representative Western blots show active Rac1 levels and their corresponding total Rac levels. B , p35/Cdk5 does not affect RasGRF2-mediated Ras activity. Transfections are as follows: 1, EV; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2+Cdk5; 5, EV+GTP. CHO cell lysates were incubated with GST-Raf1 and GST beads for 1 hr. Beads with bound active Ras were washed three times before addition of 2× SDS-sample buffer/β-mercaptoethanol and boiling for 5 min. Eluted active Ras samples were resolved by SDS-PAGE and transferred onto nitrocellulose, and Ras was detected with the monoclonal anti-ras Ab provided in the kit. Immunodetected samples are indicated next to the panels. C , CHO cells were cotransfected with the following: 1, EV; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2+Cdk5. Lysates were harvested, and equal amounts were resolved by SDS-PAGE, transferred onto nitrocellulose, and probed for the presence of phospho-MEK1/2, phospho-ERK1/2, total ERK1/2, total Rac1, p35, Cdk5, and RasGRF2 levels (panels are labeled for the detected proteins). Total Rac1 levels are similar in samples, whereas phospho-ERK1/2 levels are reduced after transfection of p35/Cdk5 (sample 3) (confirmed by the level of total ERK1/2 in this sample, which is higher than those of samples 2 and 4). Phospho-ERK1/2 levels are increased when the inactive Cdk5 is present (sample 4). The transfection of RasGRF2 causes an increase in phospho-ERK1/2 levels, whereas the transfection of p35/Cdk5 with RasGRF2 decreased this activation. The cotransfection of the inactive Cdk5 (lacking p35) causes the activated levels of ERK1/2 to return to the levels observed when only RasGRF2 was transfected. Immunodetected proteins are indicated next to the panels. D, Rac activity assays were performed to confirm that the serine 737 was indeed the phospho-site targeted by Cdk5 to mediate RasGRF2-mediated Rac activity. Tranfections are as follows: 1, EV; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2 S737A +p35+Cdk5; 5, RasGRF2 S717A +p35+Cdk5; 6, EV+GTP. The RasGRF2 S737A mutant behaved like wild-type RasGRF2; however, the RasGRF2 S717A mutant with p35 and Cdk5 showed the reduction in Rac activity demonstrating that serine 737 was the site targeted by Cdk5. E , Serine 737 phosphorylation by p35/Cdk5 reduces ERK1/2 activity. Transfections are as follows: 1, empty vector; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2 S737A +p35+Cdk5. Equal amounts were resolved by SDS-PAGE, transferred onto nitrocellulose, and probed for the presence of phospho-MEK1/2, phospho-ERK1/2, total ERK1/2, p35, Cdk5, and RasGRF2 levels. Immunodetected proteins are indicated next to the panels. The cotransfection of RasGRF2 S737A +p35+Cdk5 reversed the downregulation of ERK1/2 activity mediated by RasGRF2+p35+Cdk5, thus confirming that serine 737 is the site targeted by p35/Cdk5 to downregulate RasGRF2-mediated ERK1/2 activity.
    Figure Legend Snippet: p35/Cdk5 and RasGRF2 mediate cellular Rac activity and ERK1/2 activity. A , CHO cells were transfected with EV-GTP (lane 1), RasGRF2 (lane 2), RasGRF2+p35+Cdk5 (lane 3), RasGRF2+Cdk5 (lane4), and EV+GTP (lane5). CHO cell lysates were incubated with GST-PAK1 and GST beads for 1 hr. Beads with bound active Rac were washed three times in lysis–binding–wash buffer and eluted by addition of 2× SDS-sample buffer/β-mercaptoethanol and boiling for 5 min. Eluted, active Rac samples were resolved by SDS-PAGE, transferred onto nitrocellulose, and immunodetected using the monoclonal anti-Rac1 Ab provided in the kit at 1:1000 dilution. p35/Cdk5 reduced RasGRF2-mediated active Rac levels. EV lysate incubated with GTP before the pull-downs exhibited the highest active Rac levels (EV+GTP). Representative Western blots show active Rac1 levels and their corresponding total Rac levels. B , p35/Cdk5 does not affect RasGRF2-mediated Ras activity. Transfections are as follows: 1, EV; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2+Cdk5; 5, EV+GTP. CHO cell lysates were incubated with GST-Raf1 and GST beads for 1 hr. Beads with bound active Ras were washed three times before addition of 2× SDS-sample buffer/β-mercaptoethanol and boiling for 5 min. Eluted active Ras samples were resolved by SDS-PAGE and transferred onto nitrocellulose, and Ras was detected with the monoclonal anti-ras Ab provided in the kit. Immunodetected samples are indicated next to the panels. C , CHO cells were cotransfected with the following: 1, EV; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2+Cdk5. Lysates were harvested, and equal amounts were resolved by SDS-PAGE, transferred onto nitrocellulose, and probed for the presence of phospho-MEK1/2, phospho-ERK1/2, total ERK1/2, total Rac1, p35, Cdk5, and RasGRF2 levels (panels are labeled for the detected proteins). Total Rac1 levels are similar in samples, whereas phospho-ERK1/2 levels are reduced after transfection of p35/Cdk5 (sample 3) (confirmed by the level of total ERK1/2 in this sample, which is higher than those of samples 2 and 4). Phospho-ERK1/2 levels are increased when the inactive Cdk5 is present (sample 4). The transfection of RasGRF2 causes an increase in phospho-ERK1/2 levels, whereas the transfection of p35/Cdk5 with RasGRF2 decreased this activation. The cotransfection of the inactive Cdk5 (lacking p35) causes the activated levels of ERK1/2 to return to the levels observed when only RasGRF2 was transfected. Immunodetected proteins are indicated next to the panels. D, Rac activity assays were performed to confirm that the serine 737 was indeed the phospho-site targeted by Cdk5 to mediate RasGRF2-mediated Rac activity. Tranfections are as follows: 1, EV; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2 S737A +p35+Cdk5; 5, RasGRF2 S717A +p35+Cdk5; 6, EV+GTP. The RasGRF2 S737A mutant behaved like wild-type RasGRF2; however, the RasGRF2 S717A mutant with p35 and Cdk5 showed the reduction in Rac activity demonstrating that serine 737 was the site targeted by Cdk5. E , Serine 737 phosphorylation by p35/Cdk5 reduces ERK1/2 activity. Transfections are as follows: 1, empty vector; 2, RasGRF2; 3, RasGRF2+p35+Cdk5; 4, RasGRF2 S737A +p35+Cdk5. Equal amounts were resolved by SDS-PAGE, transferred onto nitrocellulose, and probed for the presence of phospho-MEK1/2, phospho-ERK1/2, total ERK1/2, p35, Cdk5, and RasGRF2 levels. Immunodetected proteins are indicated next to the panels. The cotransfection of RasGRF2 S737A +p35+Cdk5 reversed the downregulation of ERK1/2 activity mediated by RasGRF2+p35+Cdk5, thus confirming that serine 737 is the site targeted by p35/Cdk5 to downregulate RasGRF2-mediated ERK1/2 activity.

    Techniques Used: Activity Assay, Transfection, Incubation, Lysis, Binding Assay, SDS Page, Western Blot, Labeling, Activation Assay, Cotransfection, Mutagenesis, Plasmid Preparation

    7) Product Images from "Anti-cancer mechanisms in two murine bone marrow derived-DC subsets activated with Toll-like receptor 4 agonists"

    Article Title: Anti-cancer mechanisms in two murine bone marrow derived-DC subsets activated with Toll-like receptor 4 agonists

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1701126

    Inhibition of 4T1-GFP cells growth by TLR4 activated BMDC is partially associated with IFN-β and partially peroxynitrite. (A) Dose-dependent inhibition of type-I-Interferon production (TBK1 kinase) by Bx795 during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated. (*Note: asterisks indicate significant difference in comparison to respective 4T1-GFP growth suppression without Bx795). (B) Growth inhibition of 4T1-GFP cells by BMDC conditioned medium obtained after 24 h incubation with or without IMM or LPS in the presence or absence of Bx795. (C) Schematic diagram depicting inhibition steps in peroxynitrite pathway with indicated inhibitors. (D-F) Quantity of nitrites measured in the non-activated or TLR-4 activated BMDC conditioned media after 24 hours of incubation with different concentrations of L-NMMA (D) , FeTPPS (E) and β-Mercaptoethanol (F) . (G) Dose-dependent inhibition of NO-synthase with L-NMMA during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated. (H) Dose-dependent destruction of peroxynitrites with FeTPPS during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated. (I) Reduction of ROS by β-Mercaptoethanol during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated (* Note: asterisks indicate significant difference in comparison to respective 4T1-GFP growth suppression without inhibitor). (J) Growth inhibition of 4T1-GFP cells by BMDC conditioned medium obtained after 24 h incubation with or without IMM or LPS in the presence or absence of indicated inhibitors (L-NMMA 100μM, FeTPPS 50μM).
    Figure Legend Snippet: Inhibition of 4T1-GFP cells growth by TLR4 activated BMDC is partially associated with IFN-β and partially peroxynitrite. (A) Dose-dependent inhibition of type-I-Interferon production (TBK1 kinase) by Bx795 during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated. (*Note: asterisks indicate significant difference in comparison to respective 4T1-GFP growth suppression without Bx795). (B) Growth inhibition of 4T1-GFP cells by BMDC conditioned medium obtained after 24 h incubation with or without IMM or LPS in the presence or absence of Bx795. (C) Schematic diagram depicting inhibition steps in peroxynitrite pathway with indicated inhibitors. (D-F) Quantity of nitrites measured in the non-activated or TLR-4 activated BMDC conditioned media after 24 hours of incubation with different concentrations of L-NMMA (D) , FeTPPS (E) and β-Mercaptoethanol (F) . (G) Dose-dependent inhibition of NO-synthase with L-NMMA during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated. (H) Dose-dependent destruction of peroxynitrites with FeTPPS during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated. (I) Reduction of ROS by β-Mercaptoethanol during the coculture of 4T1-GFP with BMDC activated with IMM or LPS or non-activated (* Note: asterisks indicate significant difference in comparison to respective 4T1-GFP growth suppression without inhibitor). (J) Growth inhibition of 4T1-GFP cells by BMDC conditioned medium obtained after 24 h incubation with or without IMM or LPS in the presence or absence of indicated inhibitors (L-NMMA 100μM, FeTPPS 50μM).

    Techniques Used: Inhibition, Incubation

    8) Product Images from "Ferroptosis: A Novel Anti-tumor Action for Cisplatin"

    Article Title: Ferroptosis: A Novel Anti-tumor Action for Cisplatin

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    doi: 10.4143/crt.2016.572

    Improved anti-tumor activity was observed in combination of cisplatin and erastin. (A) A549 cells were treated with cisplatin and erastin of different concentrations as demonstrated for 48 hours. (B) HCT116 cells were treated with cisplatin and erastin of different concentrations as demonstrated for 48 hours. (C) A549 and HCT116 cells were under the treatment of cisplatin (5 μg/mL) or erastin (10 μmol/L) for 48 hours, together with z-vad-fmk (20 μmol/L) or ferrostatin-1 (Fer-1, 0.5 μmol/L), respectively. Cell viabilities were analyzed by MTT. C, cisplatin; E, erastin; F, ferrostatin-1 (Fer-1); Z, z-vad-fmk; β-ME, β-mercaptoethanol; n.s., not significant. Standard error represents three independent experiments (n=3). ** p
    Figure Legend Snippet: Improved anti-tumor activity was observed in combination of cisplatin and erastin. (A) A549 cells were treated with cisplatin and erastin of different concentrations as demonstrated for 48 hours. (B) HCT116 cells were treated with cisplatin and erastin of different concentrations as demonstrated for 48 hours. (C) A549 and HCT116 cells were under the treatment of cisplatin (5 μg/mL) or erastin (10 μmol/L) for 48 hours, together with z-vad-fmk (20 μmol/L) or ferrostatin-1 (Fer-1, 0.5 μmol/L), respectively. Cell viabilities were analyzed by MTT. C, cisplatin; E, erastin; F, ferrostatin-1 (Fer-1); Z, z-vad-fmk; β-ME, β-mercaptoethanol; n.s., not significant. Standard error represents three independent experiments (n=3). ** p

    Techniques Used: Activity Assay, MTT Assay

    Related Articles

    Irradiation:

    Article Title: Genetic correction of ?-thalassemia patient-specific iPS cells and its use in improving hemoglobin production in irradiated SCID mice
    Article Snippet: .. Human ES cells were maintained on irradiated MEF feeder cells in human ES cell medium: Knockout DMEM (Invitrogen) supplemented with 15% knockout serum replacement (Invitrogen), 5% inactivated FBS (Hyclone), 4 ng/ml bFGF (Peprotech), 10−4 M non-essential amino acids (Millipore), 10−4 M β-mercaptoethanol (Millipore), 2 mM L-glutamax (Invitrogen) and 50 μg/ml penicillin/streptomycin (Millipore). iPS cells were maintained on irradiated MEF feeder cells in DMEM/F12 (Invitrogen) supplemented with 20% knockout serum replacement (Invitrogen), 10 ng/ml bFGF (Peprotech), 10−4 M non-essential amino acids (Millipore), 10−4 M β-mercaptoethanol (Millipore), 2 mM L-glutamax (Invitrogen) and 50 μg/ml penicillin/streptomycin (Millipore). .. The plasmids pMIG containing human OCT4 , SOX2 , and KLF4 , obtained from Addgene, were transiently co-transfected with package plasmids into 293T cells using the transfect reagent Vigofect (Vigorous).

    In Vitro:

    Article Title: Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization
    Article Snippet: The zygotes were cultured in an in vitro production system using SOFaa medium supplemented with 10% fetal bovine serum (Gibco). .. The hatched blastocysts were cultured on feeder layers with the culture medium I which consisted of 90%DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 0.1 mM β-Mercaptoethanol (Chemicon), 0.1 mM nonessential amino acids, 100 IU/mL penicillin, 0.05 mg/mL streptomycin, 20 ng/mL LIF, 10 ng/mL bFGF; the culture medium II which consisted of 90%DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 0.1 mM β-Mercaptoethanol (Chemicon),0.1 mM nonessential amino acids, 100 IU/mL penicillin, 0.05 mg/mL streptomycin, 20 ng/mL LIF, 10 ng/mL bFGF, 10 ng/mL SCF; the culture medium III which consisted of 90%DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 0.1 mM β-Mercaptoethanol (Chemicon),0.1 mM nonessential amino acids, 100 IU/mL penicillin, 0.05 mg/mL streptomycin, 10 ng/mL bFGF, 10 ng/mL SCF (Table ).

    Positive Control:

    Article Title: p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons
    Article Snippet: An equal amount of EV lysate was incubated with GTP as a positive control. .. GST-bound active Rac1 and Raf were washed three times in buffer and eluted from the GST beads using 2×SDS sample buffer containing 5% β-mercaptoethanol (Sigma) and heated for 5 min.

    Homogenization:

    Article Title: RNA sequencing and proteomics approaches reveal novel deficits in the cortex of Mecp2-deficient mice, a model for Rett syndrome
    Article Snippet: .. RNA was collected using the Invitrogen™ Ambion™ PureLink™ RNA Mini Kit (Fisher Scientific) according to the manufacturer’s instructions, with the following modifications: (1) tissue was homogenized in 1 mL lysis buffer with β-mercaptoethanol (Sigma-Aldrich) in a dounce homogenizer 5 times and rested on ice for 5 min, followed by another 5 rounds of homogenization; (2) approximately 15 mg of the homogenate was removed and brought up to a final volume of 600 μL in lysis buffer with β-mercaptoethanol (Sigma-Aldrich), then re-homogenized as described above. ..

    Passaging:

    Article Title: Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization
    Article Snippet: The hatched blastocysts were cultured on feeder layers with the culture medium I which consisted of 90%DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 0.1 mM β-Mercaptoethanol (Chemicon), 0.1 mM nonessential amino acids, 100 IU/mL penicillin, 0.05 mg/mL streptomycin, 20 ng/mL LIF, 10 ng/mL bFGF; the culture medium II which consisted of 90%DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 0.1 mM β-Mercaptoethanol (Chemicon),0.1 mM nonessential amino acids, 100 IU/mL penicillin, 0.05 mg/mL streptomycin, 20 ng/mL LIF, 10 ng/mL bFGF, 10 ng/mL SCF; the culture medium III which consisted of 90%DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 0.1 mM β-Mercaptoethanol (Chemicon),0.1 mM nonessential amino acids, 100 IU/mL penicillin, 0.05 mg/mL streptomycin, 10 ng/mL bFGF, 10 ng/mL SCF (Table ). .. After the hatched blastocysts attached on the feeder layers, inner cell masses that grew well were used for passaging.

    Protease Inhibitor:

    Article Title: RNA sequencing and proteomics approaches reveal novel deficits in the cortex of Mecp2-deficient mice, a model for Rett syndrome
    Article Snippet: RNA was collected using the Invitrogen™ Ambion™ PureLink™ RNA Mini Kit (Fisher Scientific) according to the manufacturer’s instructions, with the following modifications: (1) tissue was homogenized in 1 mL lysis buffer with β-mercaptoethanol (Sigma-Aldrich) in a dounce homogenizer 5 times and rested on ice for 5 min, followed by another 5 rounds of homogenization; (2) approximately 15 mg of the homogenate was removed and brought up to a final volume of 600 μL in lysis buffer with β-mercaptoethanol (Sigma-Aldrich), then re-homogenized as described above. .. Proteins were isolated from the remaining cortical hemisphere in ice-cold lysis buffer (100 mM Tris base, pH 7.5 at room temperature, 1% (w /v ) SDS) with protease inhibitor cocktail and phosphatase inhibitor cocktail 3 (Sigma-Aldrich, product numbers P8340 and P0044, respectively) such that the final concentration was 40 mg/mL.

    Growth Inhibition Assay:

    Article Title: Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level
    Article Snippet: Paragraph title: Zone of growth inhibition assay ... A sterile disc of Whatman filter paper was placed in the center of plate and 10 μl 14 M β-mercaptoethanol (Sigma Aldrich) was applied to the center of the disk.

    Article Title: Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level
    Article Snippet: .. Zone of growth inhibition assay Assays measuring the zone of growth inhibition of different strains to β-mercaptoethanol (Sigma-Aldrich) were conducted essentially as previously described [ ]. ..

    Sonication:

    Article Title: RNA sequencing and proteomics approaches reveal novel deficits in the cortex of Mecp2-deficient mice, a model for Rett syndrome
    Article Snippet: RNA was collected using the Invitrogen™ Ambion™ PureLink™ RNA Mini Kit (Fisher Scientific) according to the manufacturer’s instructions, with the following modifications: (1) tissue was homogenized in 1 mL lysis buffer with β-mercaptoethanol (Sigma-Aldrich) in a dounce homogenizer 5 times and rested on ice for 5 min, followed by another 5 rounds of homogenization; (2) approximately 15 mg of the homogenate was removed and brought up to a final volume of 600 μL in lysis buffer with β-mercaptoethanol (Sigma-Aldrich), then re-homogenized as described above. .. Lysates were sonicated using the Model 120 Sonic Dismembrator (Fisher Scientific) for 7 s at 70% amplitude, pulse 20 s, and rest 50 s for 2 cycles.

    Blocking Assay:

    Article Title: Anti-cancer mechanisms in two murine bone marrow derived-DC subsets activated with Toll-like receptor 4 agonists
    Article Snippet: Blocking antibodies against TRAIL, FasL, TNFR1 (Biolegend, USA), ultrapure mouse rIFNβ, rTNFα (Biolegend, USA). .. DAPI, Cell Trace Violet (Invitrogen, USA), L-NMMA, β-Mercaptoethanol (Sigma, USA), FeTPPS (Calbiochem, USA), Bx795 (Invitrogen, USA).

    Article Title: Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition
    Article Snippet: 50-100 µg of proteins were heated at 70°C for 10 minutes in presence of Bolt Loading Buffer 1x supplemented with 4% β-mercaptoethanol (Sigma). .. Membranes were then incubated in blocking buffer (5% BSA or 5% milk in TBS 1x + 0.01 % Tween 20) for one hour at room temperature.

    Knock-Out:

    Article Title: Genetic correction of ?-thalassemia patient-specific iPS cells and its use in improving hemoglobin production in irradiated SCID mice
    Article Snippet: .. Human ES cells were maintained on irradiated MEF feeder cells in human ES cell medium: Knockout DMEM (Invitrogen) supplemented with 15% knockout serum replacement (Invitrogen), 5% inactivated FBS (Hyclone), 4 ng/ml bFGF (Peprotech), 10−4 M non-essential amino acids (Millipore), 10−4 M β-mercaptoethanol (Millipore), 2 mM L-glutamax (Invitrogen) and 50 μg/ml penicillin/streptomycin (Millipore). iPS cells were maintained on irradiated MEF feeder cells in DMEM/F12 (Invitrogen) supplemented with 20% knockout serum replacement (Invitrogen), 10 ng/ml bFGF (Peprotech), 10−4 M non-essential amino acids (Millipore), 10−4 M β-mercaptoethanol (Millipore), 2 mM L-glutamax (Invitrogen) and 50 μg/ml penicillin/streptomycin (Millipore). .. The plasmids pMIG containing human OCT4 , SOX2 , and KLF4 , obtained from Addgene, were transiently co-transfected with package plasmids into 293T cells using the transfect reagent Vigofect (Vigorous).

    Article Title: A time frame permissive for Protein Kinase D2 activity to direct angiogenesis in mouse embryonic stem cells
    Article Snippet: .. Cell Culture Mouse embryonic fibroblast (MEF) feeder cells were cultured in DMEM supplemented with 10%, FCS (PAA, Austria), 1% Penicillin/Streptomycin, 2 mM GlutaMax (Invitrogen, Germany), 1% Non-Essential Amino Acids (NEAA; Life-technologies, USA), 1 mM Sodium Pyruvate (Invitrogen, Germany), 1% β-Mercaptoethanol (Millipore, Germany) and 0,05 mg/ml Vitamin C (Sigma, Germany) in humidified atmosphere containing 5% CO2 at 37 °C . mESCs and miPSCs were cultured in Knockout-DMEM (KODMEM; Life-technologies), 15% FCS (Sigma, ESC-qualified), 1% Penicillin/Streptomycin, 1% GlutaMax, 1% NEAA, 1% Sodium Pyruvate, 1% β-Mercaptoethanol and 240 U/ml leukaemia inhibitory factor (LIF; Sigma, USA). .. Additionally, PD0325901 (1 μM) and GSK3 inhibitor CHIR99021 (3 μM) (Selleckchem, USA) were added to the culture medium.

    Concentration Assay:

    Article Title: RNA sequencing and proteomics approaches reveal novel deficits in the cortex of Mecp2-deficient mice, a model for Rett syndrome
    Article Snippet: RNA was collected using the Invitrogen™ Ambion™ PureLink™ RNA Mini Kit (Fisher Scientific) according to the manufacturer’s instructions, with the following modifications: (1) tissue was homogenized in 1 mL lysis buffer with β-mercaptoethanol (Sigma-Aldrich) in a dounce homogenizer 5 times and rested on ice for 5 min, followed by another 5 rounds of homogenization; (2) approximately 15 mg of the homogenate was removed and brought up to a final volume of 600 μL in lysis buffer with β-mercaptoethanol (Sigma-Aldrich), then re-homogenized as described above. .. Proteins were isolated from the remaining cortical hemisphere in ice-cold lysis buffer (100 mM Tris base, pH 7.5 at room temperature, 1% (w /v ) SDS) with protease inhibitor cocktail and phosphatase inhibitor cocktail 3 (Sigma-Aldrich, product numbers P8340 and P0044, respectively) such that the final concentration was 40 mg/mL.

    Incubation:

    Article Title: Investigating the mincing method for isolation of adipose-derived stem cells from pregnant women fat
    Article Snippet: The first step of the method was seeding P-ADSCs into a 100 mm dish (1 x106 cells/dish) containing 2% FBS/DMEM (high glucose) supplemented with 1% non-essential amino acids (NEAA) and 0.5 mM β-mercaptoethanol (Sigma) for 2 days. .. In the last step, the cells were incubated in 5% FBS/DMEM supplemented with 200 ng/mL activin A, 10 mM nicotinamide, and 10 nM exendin 4 (Sigma-Aldrich, St. Louis, MO, USA) for 7 days.

    Article Title: Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition
    Article Snippet: 50-100 µg of proteins were heated at 70°C for 10 minutes in presence of Bolt Loading Buffer 1x supplemented with 4% β-mercaptoethanol (Sigma). .. Membranes were then incubated in blocking buffer (5% BSA or 5% milk in TBS 1x + 0.01 % Tween 20) for one hour at room temperature.

    Article Title: Ethanol Cellular Defense Induce Unfolded Protein Response in Yeast
    Article Snippet: .. After overnight incubation at 28°C, 1 mL of each tube was centrifuged at 845 g for 5 min, and pellets were washed with sterile saline solution (0.9% NaCl), centrifuged and diluted to an optical density (OD600 ) of 0.15–0.2 in 250 μl of GPY medium modified with different concentrations (0–45 mM) of β-mercaptoethanol (Sigma-Aldrich). .. Growth was monitored in 96-well plates at 600 nm for 72 h in a SPECTROstar Omega instrument (BMG Labtech, Offenburg, Germany); measurements were taken every 30 min after pre-shaking for 40 s. All the experiments were carried out in triplicate under aerobic conditions and uninoculated wells for each experimental series were also included to subtract the noise signal.

    Article Title: p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons
    Article Snippet: The bound complexes were then incubated with glutathione-Sepharose beads provided for 1 hr at 4°C. .. GST-bound active Rac1 and Raf were washed three times in buffer and eluted from the GST beads using 2×SDS sample buffer containing 5% β-mercaptoethanol (Sigma) and heated for 5 min.

    Inhibition:

    Article Title: Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level
    Article Snippet: Zone of growth inhibition assay Assays measuring the zone of growth inhibition of different strains to β-mercaptoethanol (Sigma-Aldrich) were conducted essentially as previously described [ ]. .. A sterile disc of Whatman filter paper was placed in the center of plate and 10 μl 14 M β-mercaptoethanol (Sigma Aldrich) was applied to the center of the disk.

    Article Title: Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level
    Article Snippet: .. Zone of growth inhibition assay Assays measuring the zone of growth inhibition of different strains to β-mercaptoethanol (Sigma-Aldrich) were conducted essentially as previously described [ ]. ..

    Cell Culture:

    Article Title: Investigating the mincing method for isolation of adipose-derived stem cells from pregnant women fat
    Article Snippet: The first step of the method was seeding P-ADSCs into a 100 mm dish (1 x106 cells/dish) containing 2% FBS/DMEM (high glucose) supplemented with 1% non-essential amino acids (NEAA) and 0.5 mM β-mercaptoethanol (Sigma) for 2 days. .. In the second step, the cells were cultured for 7 days in 2% FBS/DMEM (high glucose) supplemented with 200 ng/mL activin A (Prospec, Rehovot, Israel), 10 mM nicotinamide (Sigma-Aldrich, St. Louis, MO, USA), 1 mM β-mercaptoethanol, 10 ng/mL basic fibroblast growth factor (bFGF, R & D Systems, Minneapolis, MN, USA), 10 ng/mL epidermal growth factor (EGF, R & D Systems, Minneapolis, MN, USA), and 25 mM glucose for 7 days.

    Article Title: Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization
    Article Snippet: .. The hatched blastocysts were cultured on feeder layers with the culture medium I which consisted of 90%DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 0.1 mM β-Mercaptoethanol (Chemicon), 0.1 mM nonessential amino acids, 100 IU/mL penicillin, 0.05 mg/mL streptomycin, 20 ng/mL LIF, 10 ng/mL bFGF; the culture medium II which consisted of 90%DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 0.1 mM β-Mercaptoethanol (Chemicon),0.1 mM nonessential amino acids, 100 IU/mL penicillin, 0.05 mg/mL streptomycin, 20 ng/mL LIF, 10 ng/mL bFGF, 10 ng/mL SCF; the culture medium III which consisted of 90%DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 0.1 mM β-Mercaptoethanol (Chemicon),0.1 mM nonessential amino acids, 100 IU/mL penicillin, 0.05 mg/mL streptomycin, 10 ng/mL bFGF, 10 ng/mL SCF (Table ). .. The bovine embryonic stem cell-like cells were also cultured in three types of culture medium which contained different factors.

    Article Title: Genetic correction of ?-thalassemia patient-specific iPS cells and its use in improving hemoglobin production in irradiated SCID mice
    Article Snippet: Paragraph title: Cell culture ... Human ES cells were maintained on irradiated MEF feeder cells in human ES cell medium: Knockout DMEM (Invitrogen) supplemented with 15% knockout serum replacement (Invitrogen), 5% inactivated FBS (Hyclone), 4 ng/ml bFGF (Peprotech), 10−4 M non-essential amino acids (Millipore), 10−4 M β-mercaptoethanol (Millipore), 2 mM L-glutamax (Invitrogen) and 50 μg/ml penicillin/streptomycin (Millipore). iPS cells were maintained on irradiated MEF feeder cells in DMEM/F12 (Invitrogen) supplemented with 20% knockout serum replacement (Invitrogen), 10 ng/ml bFGF (Peprotech), 10−4 M non-essential amino acids (Millipore), 10−4 M β-mercaptoethanol (Millipore), 2 mM L-glutamax (Invitrogen) and 50 μg/ml penicillin/streptomycin (Millipore).

    Article Title: A time frame permissive for Protein Kinase D2 activity to direct angiogenesis in mouse embryonic stem cells
    Article Snippet: .. Cell Culture Mouse embryonic fibroblast (MEF) feeder cells were cultured in DMEM supplemented with 10%, FCS (PAA, Austria), 1% Penicillin/Streptomycin, 2 mM GlutaMax (Invitrogen, Germany), 1% Non-Essential Amino Acids (NEAA; Life-technologies, USA), 1 mM Sodium Pyruvate (Invitrogen, Germany), 1% β-Mercaptoethanol (Millipore, Germany) and 0,05 mg/ml Vitamin C (Sigma, Germany) in humidified atmosphere containing 5% CO2 at 37 °C . mESCs and miPSCs were cultured in Knockout-DMEM (KODMEM; Life-technologies), 15% FCS (Sigma, ESC-qualified), 1% Penicillin/Streptomycin, 1% GlutaMax, 1% NEAA, 1% Sodium Pyruvate, 1% β-Mercaptoethanol and 240 U/ml leukaemia inhibitory factor (LIF; Sigma, USA). .. Additionally, PD0325901 (1 μM) and GSK3 inhibitor CHIR99021 (3 μM) (Selleckchem, USA) were added to the culture medium.

    BIA-KA:

    Article Title: Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition
    Article Snippet: Protein content was measured using BCA kit (Pierce) following manufacturer’s instructions. .. 50-100 µg of proteins were heated at 70°C for 10 minutes in presence of Bolt Loading Buffer 1x supplemented with 4% β-mercaptoethanol (Sigma).

    Modification:

    Article Title: Genetic correction of ?-thalassemia patient-specific iPS cells and its use in improving hemoglobin production in irradiated SCID mice
    Article Snippet: Patient's skin fibroblasts and 293T cells were maintained in Dulbecco's modified medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Hyclone). .. Human ES cells were maintained on irradiated MEF feeder cells in human ES cell medium: Knockout DMEM (Invitrogen) supplemented with 15% knockout serum replacement (Invitrogen), 5% inactivated FBS (Hyclone), 4 ng/ml bFGF (Peprotech), 10−4 M non-essential amino acids (Millipore), 10−4 M β-mercaptoethanol (Millipore), 2 mM L-glutamax (Invitrogen) and 50 μg/ml penicillin/streptomycin (Millipore). iPS cells were maintained on irradiated MEF feeder cells in DMEM/F12 (Invitrogen) supplemented with 20% knockout serum replacement (Invitrogen), 10 ng/ml bFGF (Peprotech), 10−4 M non-essential amino acids (Millipore), 10−4 M β-mercaptoethanol (Millipore), 2 mM L-glutamax (Invitrogen) and 50 μg/ml penicillin/streptomycin (Millipore).

    Article Title: Ethanol Cellular Defense Induce Unfolded Protein Response in Yeast
    Article Snippet: .. After overnight incubation at 28°C, 1 mL of each tube was centrifuged at 845 g for 5 min, and pellets were washed with sterile saline solution (0.9% NaCl), centrifuged and diluted to an optical density (OD600 ) of 0.15–0.2 in 250 μl of GPY medium modified with different concentrations (0–45 mM) of β-mercaptoethanol (Sigma-Aldrich). .. Growth was monitored in 96-well plates at 600 nm for 72 h in a SPECTROstar Omega instrument (BMG Labtech, Offenburg, Germany); measurements were taken every 30 min after pre-shaking for 40 s. All the experiments were carried out in triplicate under aerobic conditions and uninoculated wells for each experimental series were also included to subtract the noise signal.

    Article Title: A time frame permissive for Protein Kinase D2 activity to direct angiogenesis in mouse embryonic stem cells
    Article Snippet: Cell Culture Mouse embryonic fibroblast (MEF) feeder cells were cultured in DMEM supplemented with 10%, FCS (PAA, Austria), 1% Penicillin/Streptomycin, 2 mM GlutaMax (Invitrogen, Germany), 1% Non-Essential Amino Acids (NEAA; Life-technologies, USA), 1 mM Sodium Pyruvate (Invitrogen, Germany), 1% β-Mercaptoethanol (Millipore, Germany) and 0,05 mg/ml Vitamin C (Sigma, Germany) in humidified atmosphere containing 5% CO2 at 37 °C . mESCs and miPSCs were cultured in Knockout-DMEM (KODMEM; Life-technologies), 15% FCS (Sigma, ESC-qualified), 1% Penicillin/Streptomycin, 1% GlutaMax, 1% NEAA, 1% Sodium Pyruvate, 1% β-Mercaptoethanol and 240 U/ml leukaemia inhibitory factor (LIF; Sigma, USA). .. EB formation: Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen) supplemented with 10% FCS (Lonza, USA), 1% Penicillin/Streptomycin, 1% GlutaMax, 1% NEAA and freshly prepared 450 μM Monothioglycerol (Sigma) were used for differentiation.

    Western Blot:

    Article Title: Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition
    Article Snippet: Paragraph title: Protein lysates and Western Blot ... 50-100 µg of proteins were heated at 70°C for 10 minutes in presence of Bolt Loading Buffer 1x supplemented with 4% β-mercaptoethanol (Sigma).

    Article Title: p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons
    Article Snippet: GST-bound active Rac1 and Raf were washed three times in buffer and eluted from the GST beads using 2×SDS sample buffer containing 5% β-mercaptoethanol (Sigma) and heated for 5 min. .. Samples were separated using 4–20% SDS-PAGE and subjected to Western blotting using the monoclonal anti-Rac1 and Ras Ab supplied in the kit.

    Lysis:

    Article Title: RNA sequencing and proteomics approaches reveal novel deficits in the cortex of Mecp2-deficient mice, a model for Rett syndrome
    Article Snippet: .. RNA was collected using the Invitrogen™ Ambion™ PureLink™ RNA Mini Kit (Fisher Scientific) according to the manufacturer’s instructions, with the following modifications: (1) tissue was homogenized in 1 mL lysis buffer with β-mercaptoethanol (Sigma-Aldrich) in a dounce homogenizer 5 times and rested on ice for 5 min, followed by another 5 rounds of homogenization; (2) approximately 15 mg of the homogenate was removed and brought up to a final volume of 600 μL in lysis buffer with β-mercaptoethanol (Sigma-Aldrich), then re-homogenized as described above. ..

    Article Title: p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons
    Article Snippet: For the Rac assays, empty vector (EV), RasGRF2, RasGRF2+p35+Cdk5, and RasGRF2+Cdk5 were cotransfected into CHO cells, and cells were harvested in the provided lysis–binding–wash buffer. .. GST-bound active Rac1 and Raf were washed three times in buffer and eluted from the GST beads using 2×SDS sample buffer containing 5% β-mercaptoethanol (Sigma) and heated for 5 min.

    Isolation:

    Article Title: RNA sequencing and proteomics approaches reveal novel deficits in the cortex of Mecp2-deficient mice, a model for Rett syndrome
    Article Snippet: Paragraph title: RNA and protein isolation ... RNA was collected using the Invitrogen™ Ambion™ PureLink™ RNA Mini Kit (Fisher Scientific) according to the manufacturer’s instructions, with the following modifications: (1) tissue was homogenized in 1 mL lysis buffer with β-mercaptoethanol (Sigma-Aldrich) in a dounce homogenizer 5 times and rested on ice for 5 min, followed by another 5 rounds of homogenization; (2) approximately 15 mg of the homogenate was removed and brought up to a final volume of 600 μL in lysis buffer with β-mercaptoethanol (Sigma-Aldrich), then re-homogenized as described above.

    SDS Page:

    Article Title: p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons
    Article Snippet: GST-bound active Rac1 and Raf were washed three times in buffer and eluted from the GST beads using 2×SDS sample buffer containing 5% β-mercaptoethanol (Sigma) and heated for 5 min. .. Samples were separated using 4–20% SDS-PAGE and subjected to Western blotting using the monoclonal anti-Rac1 and Ras Ab supplied in the kit.

    Plasmid Preparation:

    Article Title: p35/Cyclin-Dependent Kinase 5 Phosphorylation of Ras Guanine Nucleotide Releasing Factor 2 (RasGRF2) Mediates Rac-Dependent Extracellular Signal-Regulated Kinase 1/2 Activity, Altering RasGRF2 and Microtubule-Associated Protein 1b Distribution in Neurons
    Article Snippet: For the Rac assays, empty vector (EV), RasGRF2, RasGRF2+p35+Cdk5, and RasGRF2+Cdk5 were cotransfected into CHO cells, and cells were harvested in the provided lysis–binding–wash buffer. .. GST-bound active Rac1 and Raf were washed three times in buffer and eluted from the GST beads using 2×SDS sample buffer containing 5% β-mercaptoethanol (Sigma) and heated for 5 min.

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    Millipore leukemia inhibitory factor lif millipore
    Leukemia Inhibitory Factor Lif Millipore, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/leukemia inhibitory factor lif millipore/product/Millipore
    Average 90 stars, based on 1 article reviews
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    leukemia inhibitory factor lif millipore - by Bioz Stars, 2020-01
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    97
    Millipore nmr buffer
    Transient interactions between p1–p6 and NCd domains highlighted by <t>NMR</t> data measured on the precursor and mature forms of the NCd. ( A ) Peaks of F16, N17, C18, A25, K33 amide groups in NCp7 (black), in NCp9 (purple) and in <t>NCp15</t> (red); and peaks of L57, I60 and W61 amide groups in NCp9 (purple) and in NCp15 (red), all extracted from 1 H– 15 N BEST-TROSY. ( B ) Contributions of p1–p6 (black bars), p1(light green bars) and p6 (dark green bars) to NCd chemical shift perturbations measured for the amide groups. The p1–p6 (respectively p1) contributions were calculated as the difference in chemical shifts of NCd amide groups (combined 1 H/ 15 N shifts) between NCp15 (respectively NCp9) and NCp7, the p6 contributions were calculated as the difference in chemical shifts of NCd amide groups (combined 1 H/ 15 N shifts) between NCp15 and NCp9. The delimitation of the different regions in NCd is indicated as a drawing placed above the graphs using the color code of Figure 1A . ( C ) 2D NOESY (150 ms) measured in D 2 O on NCp9 (in purple) showing NOEs between proton Hϵ3 of W37 and protons of methyl groups of L57 and I60, the same region of a 2D NOESY recorded in the same conditions on NCp7 is displayed in black. ( D ) Schematic summarizing the interactions between NCd, p1 and p6 highlighted by the NMR chemical shifts and the dynamics analysis of the NCd in NCp7, NCp9 and NCp15.
    Nmr Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nmr buffer/product/Millipore
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    93
    Millipore ksr medium
    LCD shows direct differentiation outcomes of <t>H9</t> cells seeded at different densities A-D , Undifferentiated H9 cells with localized high cell density were subjected to IF using anti-PAX6 (A and D) and anti-OCT4 (B and D) antibodies. E-L , H9 cells seeded at low density (8×10 3 cells/ cm 2 ) (E-H) and high density (1×10 4 cells/ cm 2 ) (I-L) were treated with <t>KSR</t> and N2 medium supplemented with noggin and SB431542 for 5 days. The cells were then subjected to the IF assay using anti-PAX6 antibody (green, E, H, I and L) and anti-OCT4 antibody (red, F, H, J and L) in the H9-derived cells. M, The areas of the signals in the OCT4, PAX6 and DAPI channels were calculated using Image J software. The ratios of the OCT4 and PAX6 area to DAPI area are shown (X±SD, n=8; *, P
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    Image Search Results


    Transient interactions between p1–p6 and NCd domains highlighted by NMR data measured on the precursor and mature forms of the NCd. ( A ) Peaks of F16, N17, C18, A25, K33 amide groups in NCp7 (black), in NCp9 (purple) and in NCp15 (red); and peaks of L57, I60 and W61 amide groups in NCp9 (purple) and in NCp15 (red), all extracted from 1 H– 15 N BEST-TROSY. ( B ) Contributions of p1–p6 (black bars), p1(light green bars) and p6 (dark green bars) to NCd chemical shift perturbations measured for the amide groups. The p1–p6 (respectively p1) contributions were calculated as the difference in chemical shifts of NCd amide groups (combined 1 H/ 15 N shifts) between NCp15 (respectively NCp9) and NCp7, the p6 contributions were calculated as the difference in chemical shifts of NCd amide groups (combined 1 H/ 15 N shifts) between NCp15 and NCp9. The delimitation of the different regions in NCd is indicated as a drawing placed above the graphs using the color code of Figure 1A . ( C ) 2D NOESY (150 ms) measured in D 2 O on NCp9 (in purple) showing NOEs between proton Hϵ3 of W37 and protons of methyl groups of L57 and I60, the same region of a 2D NOESY recorded in the same conditions on NCp7 is displayed in black. ( D ) Schematic summarizing the interactions between NCd, p1 and p6 highlighted by the NMR chemical shifts and the dynamics analysis of the NCd in NCp7, NCp9 and NCp15.

    Journal: Nucleic Acids Research

    Article Title: Modulation of the HIV nucleocapsid dynamics finely tunes its RNA-binding properties during virion genesis

    doi: 10.1093/nar/gky612

    Figure Lengend Snippet: Transient interactions between p1–p6 and NCd domains highlighted by NMR data measured on the precursor and mature forms of the NCd. ( A ) Peaks of F16, N17, C18, A25, K33 amide groups in NCp7 (black), in NCp9 (purple) and in NCp15 (red); and peaks of L57, I60 and W61 amide groups in NCp9 (purple) and in NCp15 (red), all extracted from 1 H– 15 N BEST-TROSY. ( B ) Contributions of p1–p6 (black bars), p1(light green bars) and p6 (dark green bars) to NCd chemical shift perturbations measured for the amide groups. The p1–p6 (respectively p1) contributions were calculated as the difference in chemical shifts of NCd amide groups (combined 1 H/ 15 N shifts) between NCp15 (respectively NCp9) and NCp7, the p6 contributions were calculated as the difference in chemical shifts of NCd amide groups (combined 1 H/ 15 N shifts) between NCp15 and NCp9. The delimitation of the different regions in NCd is indicated as a drawing placed above the graphs using the color code of Figure 1A . ( C ) 2D NOESY (150 ms) measured in D 2 O on NCp9 (in purple) showing NOEs between proton Hϵ3 of W37 and protons of methyl groups of L57 and I60, the same region of a 2D NOESY recorded in the same conditions on NCp7 is displayed in black. ( D ) Schematic summarizing the interactions between NCd, p1 and p6 highlighted by the NMR chemical shifts and the dynamics analysis of the NCd in NCp7, NCp9 and NCp15.

    Article Snippet: The fractions containing NCp15 were pooled, concentrated and dialyzed against the NMR buffer (25 mM deutered sodium acetate pH 6.0, 25 mM NaCl, 0.1 mM ZnCl2 and 0.1 mM β-mercaptoethanol) using an amicon unit of 3 kDa (Millipore).

    Techniques: Nuclear Magnetic Resonance, Mass Spectrometry

    Structural and dynamic characterization of NCp15 from NMR chemical shifts and 15 N relaxation data. ( A ) Schematic of full-length Gag, ( B ) Sequence of the HIV-1 C-terminal domain of Gag (NCp15). The first cleavage by the HIV-1 protease first liberates the NCp15 protein, then NCp9 and finally the mature form of NCd called NCp7. The dashed line represents the two sites of protease cleavage present in NCp15. Residues are coloured in grey for residues in the N-terminal part of NCd, green for those in the zinc knuckles and orange for residues in the linker between the two zinc knuckles of NCd and purple for the C-terminal domain of NCd. Due to a limitation in space, the sequence of p6 is not drawn linearly, but this does not represent a fold back of p6 on itself, ( C ) Secondary structural propensities calculated by the SSP program ( 58 ). C α , C β , CO and H α were used as input data for the calculation of the SSP score. Positive values indicate the amount of α-helical conformation present along the sequence whereas negative values indicate extended or β-strand conformations. ( D ) 15 N–{ 1 H} NOE (HetNOE) values are indicative of the magnitude of local subnanosecond motions (high values: restricted motions; low values: high-amplitude motions). ( E ) Spectral densities J(0) extracted by spectral density mapping from 15 N relaxation data (T1, T2, HetNOE) of NCp15. J(0) values are indicative of slow overall and segmental tumbling motions present in NCp15. The boxes in grey indicated the four regions of p1–p6 domains showing significant secondary structure propensities. (F) Differences of HetNOE within the NCd between NCp15 and NCp7 to probe the change in restrictions of motions of the backbone of the precursor and mature forms of the NCd.

    Journal: Nucleic Acids Research

    Article Title: Modulation of the HIV nucleocapsid dynamics finely tunes its RNA-binding properties during virion genesis

    doi: 10.1093/nar/gky612

    Figure Lengend Snippet: Structural and dynamic characterization of NCp15 from NMR chemical shifts and 15 N relaxation data. ( A ) Schematic of full-length Gag, ( B ) Sequence of the HIV-1 C-terminal domain of Gag (NCp15). The first cleavage by the HIV-1 protease first liberates the NCp15 protein, then NCp9 and finally the mature form of NCd called NCp7. The dashed line represents the two sites of protease cleavage present in NCp15. Residues are coloured in grey for residues in the N-terminal part of NCd, green for those in the zinc knuckles and orange for residues in the linker between the two zinc knuckles of NCd and purple for the C-terminal domain of NCd. Due to a limitation in space, the sequence of p6 is not drawn linearly, but this does not represent a fold back of p6 on itself, ( C ) Secondary structural propensities calculated by the SSP program ( 58 ). C α , C β , CO and H α were used as input data for the calculation of the SSP score. Positive values indicate the amount of α-helical conformation present along the sequence whereas negative values indicate extended or β-strand conformations. ( D ) 15 N–{ 1 H} NOE (HetNOE) values are indicative of the magnitude of local subnanosecond motions (high values: restricted motions; low values: high-amplitude motions). ( E ) Spectral densities J(0) extracted by spectral density mapping from 15 N relaxation data (T1, T2, HetNOE) of NCp15. J(0) values are indicative of slow overall and segmental tumbling motions present in NCp15. The boxes in grey indicated the four regions of p1–p6 domains showing significant secondary structure propensities. (F) Differences of HetNOE within the NCd between NCp15 and NCp7 to probe the change in restrictions of motions of the backbone of the precursor and mature forms of the NCd.

    Article Snippet: The fractions containing NCp15 were pooled, concentrated and dialyzed against the NMR buffer (25 mM deutered sodium acetate pH 6.0, 25 mM NaCl, 0.1 mM ZnCl2 and 0.1 mM β-mercaptoethanol) using an amicon unit of 3 kDa (Millipore).

    Techniques: Nuclear Magnetic Resonance, Sequencing

    NMR and ITC analysis of NCd/SL3 complexes. ( A ) Chemical shift perturbations upon SL3 binding for NCp7 (black bars), NCp9 (purple bars) and NCp15 (red bars) observed at the level of the NC domain. The delimitation of the different regions in NCd is indicated as a drawing placed above the graphs using the color code of Figure 1A . Stars indicate that we do not have the data for the three complexes due to peak superposition. ( B ) NMR chemical shift perturbations upon SL3 binding measured for NCp15 as a function of the sequence of NCp15. The color code for residues is that of Figure 1 . ( C ) Thermodynamic components (ΔG: free energy, ΔH: enthalpy and –TΔS: entropy) displayed as bars extracted from the calorimetric titrations of SL3 RNA with NCp7 (in black), NCp9 (in purple) and NCp15 (in red). Values are reported as means ± standard error. The uncertainties on the fitted parameters were estimated from the data spread and from the uncertainty of the protein concentration determination (5%) ( 62 ).

    Journal: Nucleic Acids Research

    Article Title: Modulation of the HIV nucleocapsid dynamics finely tunes its RNA-binding properties during virion genesis

    doi: 10.1093/nar/gky612

    Figure Lengend Snippet: NMR and ITC analysis of NCd/SL3 complexes. ( A ) Chemical shift perturbations upon SL3 binding for NCp7 (black bars), NCp9 (purple bars) and NCp15 (red bars) observed at the level of the NC domain. The delimitation of the different regions in NCd is indicated as a drawing placed above the graphs using the color code of Figure 1A . Stars indicate that we do not have the data for the three complexes due to peak superposition. ( B ) NMR chemical shift perturbations upon SL3 binding measured for NCp15 as a function of the sequence of NCp15. The color code for residues is that of Figure 1 . ( C ) Thermodynamic components (ΔG: free energy, ΔH: enthalpy and –TΔS: entropy) displayed as bars extracted from the calorimetric titrations of SL3 RNA with NCp7 (in black), NCp9 (in purple) and NCp15 (in red). Values are reported as means ± standard error. The uncertainties on the fitted parameters were estimated from the data spread and from the uncertainty of the protein concentration determination (5%) ( 62 ).

    Article Snippet: The fractions containing NCp15 were pooled, concentrated and dialyzed against the NMR buffer (25 mM deutered sodium acetate pH 6.0, 25 mM NaCl, 0.1 mM ZnCl2 and 0.1 mM β-mercaptoethanol) using an amicon unit of 3 kDa (Millipore).

    Techniques: Nuclear Magnetic Resonance, Binding Assay, Sequencing, Protein Concentration

    LCD shows direct differentiation outcomes of H9 cells seeded at different densities A-D , Undifferentiated H9 cells with localized high cell density were subjected to IF using anti-PAX6 (A and D) and anti-OCT4 (B and D) antibodies. E-L , H9 cells seeded at low density (8×10 3 cells/ cm 2 ) (E-H) and high density (1×10 4 cells/ cm 2 ) (I-L) were treated with KSR and N2 medium supplemented with noggin and SB431542 for 5 days. The cells were then subjected to the IF assay using anti-PAX6 antibody (green, E, H, I and L) and anti-OCT4 antibody (red, F, H, J and L) in the H9-derived cells. M, The areas of the signals in the OCT4, PAX6 and DAPI channels were calculated using Image J software. The ratios of the OCT4 and PAX6 area to DAPI area are shown (X±SD, n=8; *, P

    Journal: Biochemical and biophysical research communications

    Article Title: Synergistic contribution of SMAD signaling blockade and high localized cell density in the differentiation of neuroectoderm from H9 cells

    doi: 10.1016/j.bbrc.2014.08.137

    Figure Lengend Snippet: LCD shows direct differentiation outcomes of H9 cells seeded at different densities A-D , Undifferentiated H9 cells with localized high cell density were subjected to IF using anti-PAX6 (A and D) and anti-OCT4 (B and D) antibodies. E-L , H9 cells seeded at low density (8×10 3 cells/ cm 2 ) (E-H) and high density (1×10 4 cells/ cm 2 ) (I-L) were treated with KSR and N2 medium supplemented with noggin and SB431542 for 5 days. The cells were then subjected to the IF assay using anti-PAX6 antibody (green, E, H, I and L) and anti-OCT4 antibody (red, F, H, J and L) in the H9-derived cells. M, The areas of the signals in the OCT4, PAX6 and DAPI channels were calculated using Image J software. The ratios of the OCT4 and PAX6 area to DAPI area are shown (X±SD, n=8; *, P

    Article Snippet: Briefly, H9 cells were cultured on MEFs in KSR medium (DMEM/F12, 20 % KSR, 0.1 mM β-mercaptoethanol, 10 ng/ml of FGF-2) and disaggregated using accutase (Millipore, Billerica, MA, USA) for 20 min, washed with KSR medium and pre-plated on gelatin-coated 6-well plates for 1 h at 37 °C in the presence of the ROCK inhibitor (Y-27632) to remove MEFs.

    Techniques: Derivative Assay, Software

    Synergistic contribution of SMAD signaling blockers and localized high cell density in NE differentiation A-F, Five days after the cell-clump-based differentiation of NE in KSR and N2 medium with (D-F) or without (A-C) SMAD signaling blockers, H9-derived cells were subjected to the IF assay using anti-PAX6 antibody (green, A-D). The nuclei were stained using DAPI (blue, C and D). The micrographs were divided into 20 (5×4) squares as indicated (C and D). E-F , The number of total cells and PAX6-positive cells in each square was quantified using Image J software. The ratio of PAX6-positive cells to total cells in each square was determined. The squares with equivalent ratios were binned together. The ratios of PAX6-positive cells to total cells in each bin are shown (F, X±SD, **P

    Journal: Biochemical and biophysical research communications

    Article Title: Synergistic contribution of SMAD signaling blockade and high localized cell density in the differentiation of neuroectoderm from H9 cells

    doi: 10.1016/j.bbrc.2014.08.137

    Figure Lengend Snippet: Synergistic contribution of SMAD signaling blockers and localized high cell density in NE differentiation A-F, Five days after the cell-clump-based differentiation of NE in KSR and N2 medium with (D-F) or without (A-C) SMAD signaling blockers, H9-derived cells were subjected to the IF assay using anti-PAX6 antibody (green, A-D). The nuclei were stained using DAPI (blue, C and D). The micrographs were divided into 20 (5×4) squares as indicated (C and D). E-F , The number of total cells and PAX6-positive cells in each square was quantified using Image J software. The ratio of PAX6-positive cells to total cells in each square was determined. The squares with equivalent ratios were binned together. The ratios of PAX6-positive cells to total cells in each bin are shown (F, X±SD, **P

    Article Snippet: Briefly, H9 cells were cultured on MEFs in KSR medium (DMEM/F12, 20 % KSR, 0.1 mM β-mercaptoethanol, 10 ng/ml of FGF-2) and disaggregated using accutase (Millipore, Billerica, MA, USA) for 20 min, washed with KSR medium and pre-plated on gelatin-coated 6-well plates for 1 h at 37 °C in the presence of the ROCK inhibitor (Y-27632) to remove MEFs.

    Techniques: Derivative Assay, Staining, Software