Structured Review

Millipore hla dr2
( A ) SDS-PAGE analysis (12%) of detergent solubilized <t>HLA-DR2</t> (DRB5*0101/DRB1*1501; left) or HLA-DR4 (DRB1*0401; right) where 5–8×10 9 of the Epstein Barr virus (EBV)-transformed B cell line HTC-LAN and BSM were lysed, respectively, and their affinity was purified using the immobilized monoclonal antibody L243. After concentration on a 30-kDa membrane, 670–700 g of the human leukocyte antigen (HLA)-II were obtained and stored in phosphate buffered saline at a concentration of 0.5 mg/mL. The purity of HLA-DR2 and HLA-DR4 were about 85%. ( B ) Competition binding of the MBP 82–98 peptide analogues in 10-fold, 20-fold, and 50-fold excess of AMCA-labeled MBP 83–99 , which is specific to the HLA-DR2b chain, was used as the peptide competitor, and ( C ) AMCA-labeled HA 306–318 , which is specific to the HLA-DR4, was used as the peptide competitor. All of the experiments were in triplicates, where ** p
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Images

1) Product Images from "Design of Linear and Cyclic Mutant Analogues of Dirucotide Peptide (MBP82–98) against Multiple Sclerosis: Conformational and Binding Studies to MHC Class II"

Article Title: Design of Linear and Cyclic Mutant Analogues of Dirucotide Peptide (MBP82–98) against Multiple Sclerosis: Conformational and Binding Studies to MHC Class II

Journal: Brain Sciences

doi: 10.3390/brainsci8120213

( A ) SDS-PAGE analysis (12%) of detergent solubilized HLA-DR2 (DRB5*0101/DRB1*1501; left) or HLA-DR4 (DRB1*0401; right) where 5–8×10 9 of the Epstein Barr virus (EBV)-transformed B cell line HTC-LAN and BSM were lysed, respectively, and their affinity was purified using the immobilized monoclonal antibody L243. After concentration on a 30-kDa membrane, 670–700 g of the human leukocyte antigen (HLA)-II were obtained and stored in phosphate buffered saline at a concentration of 0.5 mg/mL. The purity of HLA-DR2 and HLA-DR4 were about 85%. ( B ) Competition binding of the MBP 82–98 peptide analogues in 10-fold, 20-fold, and 50-fold excess of AMCA-labeled MBP 83–99 , which is specific to the HLA-DR2b chain, was used as the peptide competitor, and ( C ) AMCA-labeled HA 306–318 , which is specific to the HLA-DR4, was used as the peptide competitor. All of the experiments were in triplicates, where ** p
Figure Legend Snippet: ( A ) SDS-PAGE analysis (12%) of detergent solubilized HLA-DR2 (DRB5*0101/DRB1*1501; left) or HLA-DR4 (DRB1*0401; right) where 5–8×10 9 of the Epstein Barr virus (EBV)-transformed B cell line HTC-LAN and BSM were lysed, respectively, and their affinity was purified using the immobilized monoclonal antibody L243. After concentration on a 30-kDa membrane, 670–700 g of the human leukocyte antigen (HLA)-II were obtained and stored in phosphate buffered saline at a concentration of 0.5 mg/mL. The purity of HLA-DR2 and HLA-DR4 were about 85%. ( B ) Competition binding of the MBP 82–98 peptide analogues in 10-fold, 20-fold, and 50-fold excess of AMCA-labeled MBP 83–99 , which is specific to the HLA-DR2b chain, was used as the peptide competitor, and ( C ) AMCA-labeled HA 306–318 , which is specific to the HLA-DR4, was used as the peptide competitor. All of the experiments were in triplicates, where ** p

Techniques Used: SDS Page, Transformation Assay, Purification, Concentration Assay, Binding Assay, Labeling

2) Product Images from "Splice Variants of Perlucin from Haliotis laevigata Modulate the Crystallisation of CaCO3"

Article Title: Splice Variants of Perlucin from Haliotis laevigata Modulate the Crystallisation of CaCO3

Journal: PLoS ONE

doi: 10.1371/journal.pone.0097126

Amino acid sequences of Perlucin from H. laevigata . Perlucin splice variants (Perlucin-R0, Perlucin-R5, Perlucin-R8) are indicated as grey bars above the sequence. The following characteristics of the proteins are marked: Amino acid exchanges in Perlucin-R0 (M89I, V129D, R149L), signal peptide, C-type lectin domain [60] , repeat units (light blue bars). Peptides identified from 2D electrophoresis spots ( Figure 3 ) by MALDI-ToF MS (black bars) or ESI-MS (red bar). *Predicted glycosylation and phosphorylation (NetPhos [50] ) sites.
Figure Legend Snippet: Amino acid sequences of Perlucin from H. laevigata . Perlucin splice variants (Perlucin-R0, Perlucin-R5, Perlucin-R8) are indicated as grey bars above the sequence. The following characteristics of the proteins are marked: Amino acid exchanges in Perlucin-R0 (M89I, V129D, R149L), signal peptide, C-type lectin domain [60] , repeat units (light blue bars). Peptides identified from 2D electrophoresis spots ( Figure 3 ) by MALDI-ToF MS (black bars) or ESI-MS (red bar). *Predicted glycosylation and phosphorylation (NetPhos [50] ) sites.

Techniques Used: Sequencing, Two-Dimensional Gel Electrophoresis, Mass Spectrometry

Electrophoretic analysis of recombinant and native Perlucin preparations. A) Western blot of cell culture supernatants derived from COS-7 cells ectopically over expressing the indicated Strep-tagged recombinant Perlucins, which were detected using a polyclonal anti-Strep-tag antibody as described in the Methods section. B) SDS-PAGE of native Perlucin purified from abalone shell of H. laevigata , stained with Coomassie brilliant blue showed one distinct band at approx. 25 kDa, one at 20 kDa, and one at approx. 15 kDa. C) 2D electrophoresis of native Perlucin purified from abalone shell of H. laevigata , stained with Coomassie Brilliant Blue. The indicated spots were cut out and analysed by MALDI-ToF MS as described in the Methods section. Spot 7 was used as control.
Figure Legend Snippet: Electrophoretic analysis of recombinant and native Perlucin preparations. A) Western blot of cell culture supernatants derived from COS-7 cells ectopically over expressing the indicated Strep-tagged recombinant Perlucins, which were detected using a polyclonal anti-Strep-tag antibody as described in the Methods section. B) SDS-PAGE of native Perlucin purified from abalone shell of H. laevigata , stained with Coomassie brilliant blue showed one distinct band at approx. 25 kDa, one at 20 kDa, and one at approx. 15 kDa. C) 2D electrophoresis of native Perlucin purified from abalone shell of H. laevigata , stained with Coomassie Brilliant Blue. The indicated spots were cut out and analysed by MALDI-ToF MS as described in the Methods section. Spot 7 was used as control.

Techniques Used: Recombinant, Western Blot, Cell Culture, Derivative Assay, Expressing, Strep-tag, SDS Page, Purification, Staining, Two-Dimensional Gel Electrophoresis, Mass Spectrometry

3) Product Images from "Effect of Uncaria tomentosa extract on purinergic enzyme activities in lymphocytes of rats submitted to experimental adjuvant arthritis model"

Article Title: Effect of Uncaria tomentosa extract on purinergic enzyme activities in lymphocytes of rats submitted to experimental adjuvant arthritis model

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/s12906-015-0694-4

ATP ( a ) and ADP ( b ) hydrolysis in lymphocytes of Complete Freund’s Adjuvant (CFA)- induced arthritis rats and treated for 45 days with Uncaria tomentosa extract in the dose of 150 mg/kg, 2 times/day. Enzyme specific activities are reported as nmol of Pi released/min/mg of protein. Groups: C (control), E (extract), A (arthritis) and A + E (arthritis + extract). Bars represent mean ± S.E.M. ( a,b ) Indicates a significant P
Figure Legend Snippet: ATP ( a ) and ADP ( b ) hydrolysis in lymphocytes of Complete Freund’s Adjuvant (CFA)- induced arthritis rats and treated for 45 days with Uncaria tomentosa extract in the dose of 150 mg/kg, 2 times/day. Enzyme specific activities are reported as nmol of Pi released/min/mg of protein. Groups: C (control), E (extract), A (arthritis) and A + E (arthritis + extract). Bars represent mean ± S.E.M. ( a,b ) Indicates a significant P

Techniques Used:

4) Product Images from "Method for simultaneous analysis of eight analogues of vitamin D using liquid chromatography tandem mass spectrometry"

Article Title: Method for simultaneous analysis of eight analogues of vitamin D using liquid chromatography tandem mass spectrometry

Journal: Chemistry Central Journal

doi: 10.1186/1752-153X-6-112

Typical chromatogram showing vitamin D analogues [1 ST period analytes: Stanozolol-d3-RT = 2.96, 1α25(OH) 2 D2-RT = 5.49, 1α25(OH) 2 D3-RT = 6.74; 2 nd period analytes: 7αC4-RT = 9.51, 25OHD3-RT = 10.86, 3-epi-25OHD3-RT = 11.21, 25OHD2-RT = 11.46 and 3-epi-25OHD2-RT = 11.71; 3 RD period analytes: vitamin D3-RT = 14.87 and vitamin D2-RT = 15.38].
Figure Legend Snippet: Typical chromatogram showing vitamin D analogues [1 ST period analytes: Stanozolol-d3-RT = 2.96, 1α25(OH) 2 D2-RT = 5.49, 1α25(OH) 2 D3-RT = 6.74; 2 nd period analytes: 7αC4-RT = 9.51, 25OHD3-RT = 10.86, 3-epi-25OHD3-RT = 11.21, 25OHD2-RT = 11.46 and 3-epi-25OHD2-RT = 11.71; 3 RD period analytes: vitamin D3-RT = 14.87 and vitamin D2-RT = 15.38].

Techniques Used:

5) Product Images from "Square Wave Voltammetry of TNT at Gold Electrodes Modified with Self-Assembled Monolayers Containing Aromatic Structures"

Article Title: Square Wave Voltammetry of TNT at Gold Electrodes Modified with Self-Assembled Monolayers Containing Aromatic Structures

Journal: PLoS ONE

doi: 10.1371/journal.pone.0115966

Experimental details. A. Chemical structures for biphenyl-4-thiol (Biphenyl), 4-(phenylethynyl)benzenethiol (OPE) and undecane-1-thiol (C11) used to from SAMs on gold electrodes. B. The electrochemical setup.
Figure Legend Snippet: Experimental details. A. Chemical structures for biphenyl-4-thiol (Biphenyl), 4-(phenylethynyl)benzenethiol (OPE) and undecane-1-thiol (C11) used to from SAMs on gold electrodes. B. The electrochemical setup.

Techniques Used:

6) Product Images from "Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum"

Article Title: Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum

Journal: Nature Communications

doi: 10.1038/ncomms10111

In vitro binding and functional validation of artemisinin targets. ( a ) Artemisinin specifically interacts with OAT, PyrK, LDH, SpdSyn, SAMS and TCTP as the unlabelled artesunate (25 ×) can compete with the AP1 binding. Heat denaturation reduces the AP1 -labelling level of OAT, suggesting that the interaction of artemisinin with OAT is activity based. ( b ) Dose-dependent labelling of OAT with AP1 (4 h treatment). ( c ) Time-dependent labelling of OAT with AP1 . ( d ) The interaction of artemisinin with OAT may involve thiol and amine groups as IAA (blocking thiol, 30 mM) and NEM (blocking amine, 10 mM) pretreatment (20 min) can reduce binding. ( e , f ) Activated artesunate inhibits the activities of PyrK ( e ) and LDH ( f ) in vitro . Δ, heat denaturation; IAA, iodoacetamide; NEM, N -ethylmaleimide; Conc., concentration. Error bars represent s.d. in three independent replicates in e and f . Full-gel images for panels a – d are shown in Supplementary Fig. 13 .
Figure Legend Snippet: In vitro binding and functional validation of artemisinin targets. ( a ) Artemisinin specifically interacts with OAT, PyrK, LDH, SpdSyn, SAMS and TCTP as the unlabelled artesunate (25 ×) can compete with the AP1 binding. Heat denaturation reduces the AP1 -labelling level of OAT, suggesting that the interaction of artemisinin with OAT is activity based. ( b ) Dose-dependent labelling of OAT with AP1 (4 h treatment). ( c ) Time-dependent labelling of OAT with AP1 . ( d ) The interaction of artemisinin with OAT may involve thiol and amine groups as IAA (blocking thiol, 30 mM) and NEM (blocking amine, 10 mM) pretreatment (20 min) can reduce binding. ( e , f ) Activated artesunate inhibits the activities of PyrK ( e ) and LDH ( f ) in vitro . Δ, heat denaturation; IAA, iodoacetamide; NEM, N -ethylmaleimide; Conc., concentration. Error bars represent s.d. in three independent replicates in e and f . Full-gel images for panels a – d are shown in Supplementary Fig. 13 .

Techniques Used: In Vitro, Binding Assay, Functional Assay, Activity Assay, Blocking Assay, Concentration Assay

7) Product Images from "Interference of Quorum Sensing by Delftia sp. VM4 Depends on the Activity of a Novel N-Acylhomoserine Lactone-Acylase"

Article Title: Interference of Quorum Sensing by Delftia sp. VM4 Depends on the Activity of a Novel N-Acylhomoserine Lactone-Acylase

Journal: PLoS ONE

doi: 10.1371/journal.pone.0138034

Characterization of AHL-acylase of Delftia sp. VM4. (A) Silver stained 12% SDS-PAGE during purification of AHL acylase from Delftia sp. VM4. Lane: M, PMWH- protein marker; 1, ammonium sulphate precipitates; 2, eluate from DEAE-sepharose column; 3, eluate from Sephadex column. (B) Microtitre plate assay for the AHL degrading activity with 0.05 mM HHL of the renatured gel slices (bands i-vi) from the SDS-PAGE was assayed using C . violaceum CV026 biosensor based bioassay. HHL (0.05 mM) was loaded as the control (well-c). HPLC profile of (C) HHL without purified AHL acylase, (D) HHL degradation by purified AHL acylase, (E) EI-MS analysis of degraded product (HSL) from HHL.
Figure Legend Snippet: Characterization of AHL-acylase of Delftia sp. VM4. (A) Silver stained 12% SDS-PAGE during purification of AHL acylase from Delftia sp. VM4. Lane: M, PMWH- protein marker; 1, ammonium sulphate precipitates; 2, eluate from DEAE-sepharose column; 3, eluate from Sephadex column. (B) Microtitre plate assay for the AHL degrading activity with 0.05 mM HHL of the renatured gel slices (bands i-vi) from the SDS-PAGE was assayed using C . violaceum CV026 biosensor based bioassay. HHL (0.05 mM) was loaded as the control (well-c). HPLC profile of (C) HHL without purified AHL acylase, (D) HHL degradation by purified AHL acylase, (E) EI-MS analysis of degraded product (HSL) from HHL.

Techniques Used: Staining, SDS Page, Purification, Marker, Activity Assay, High Performance Liquid Chromatography, Mass Spectrometry

8) Product Images from "Localization of tamoxifen in human breast cancer tumors by MALDI mass spectrometry imaging"

Article Title: Localization of tamoxifen in human breast cancer tumors by MALDI mass spectrometry imaging

Journal: Clinical and Translational Medicine

doi: 10.1186/s40169-016-0090-9

Ionization characteristics of tamoxifen (0.1 mg/mL in water) as measured with 3.5 mg/mL CHCA on a stainless steel MALDI target plate. a A full mass spectrum of tamoxifen obtained at 60,000 resolution using the Orbitrap mass analyzer and b a tandem mass spectrum of tamoxifen isolating the m/z 372.23 and CID fragmented in the linear ion trap mass analyzer
Figure Legend Snippet: Ionization characteristics of tamoxifen (0.1 mg/mL in water) as measured with 3.5 mg/mL CHCA on a stainless steel MALDI target plate. a A full mass spectrum of tamoxifen obtained at 60,000 resolution using the Orbitrap mass analyzer and b a tandem mass spectrum of tamoxifen isolating the m/z 372.23 and CID fragmented in the linear ion trap mass analyzer

Techniques Used:

9) Product Images from "Macrophage receptors of polysaccharide isolated from a marine filamentous fungus Phoma herbarum YS4108"

Article Title: Macrophage receptors of polysaccharide isolated from a marine filamentous fungus Phoma herbarum YS4108

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2009.93

Polysaccharide labeling with fluoresceinamine. CDAP-activated YCP reacted with fluoresceinamine for 18 h at room temperature. The mixtures were fractionated on a Sephacryl S-400 column, concentration (μg/mL) of fluoresceinamine (▴) and YCP (□) in each fraction were determined. n =3.
Figure Legend Snippet: Polysaccharide labeling with fluoresceinamine. CDAP-activated YCP reacted with fluoresceinamine for 18 h at room temperature. The mixtures were fractionated on a Sephacryl S-400 column, concentration (μg/mL) of fluoresceinamine (▴) and YCP (□) in each fraction were determined. n =3.

Techniques Used: Labeling, Concentration Assay

10) Product Images from "Simultaneous Determination of Salidroside and Its Aglycone Metabolite p-Tyrosol in Rat Plasma by Liquid Chromatography-Tandem Mass Spectrometry"

Article Title: Simultaneous Determination of Salidroside and Its Aglycone Metabolite p-Tyrosol in Rat Plasma by Liquid Chromatography-Tandem Mass Spectrometry

Journal: Molecules

doi: 10.3390/molecules17044733

MRM chromatograms of salidroside, p -tyrosol and IS in ( a ) a blank rat plasma sample; ( b ) a blank rat plasma spiked with salidroside (500 ng·mL −1 ), p -tyrosol (100 ng·mL −1 ) and IS (200 ng·mL −1 ); ( c ) a rat plasma sample collected 5 min after i.v. administration of salidroside (50 mg/kg) with IS (200 ng·mL −1 ); ( d ) a rat plasma sample collected 5 min after i.g. administration of salidroside (100 mg/kg) with IS (200 ng·mL −1 ); ( e ) a rat plasma sample collected 30 min after i.g. administration of salidroside (100 mg/kg) with IS (200 ng·mL −1 ); ( f ) a rat plasma sample collected 2 h after i.g. administration of salidroside (100 mg/kg) with IS (200 ng·mL −1 ); ( g ) a rat plasma sample collected 4 h after i.g. administration of salidroside (100 mg/kg) with IS (200 ng·mL −1 ).
Figure Legend Snippet: MRM chromatograms of salidroside, p -tyrosol and IS in ( a ) a blank rat plasma sample; ( b ) a blank rat plasma spiked with salidroside (500 ng·mL −1 ), p -tyrosol (100 ng·mL −1 ) and IS (200 ng·mL −1 ); ( c ) a rat plasma sample collected 5 min after i.v. administration of salidroside (50 mg/kg) with IS (200 ng·mL −1 ); ( d ) a rat plasma sample collected 5 min after i.g. administration of salidroside (100 mg/kg) with IS (200 ng·mL −1 ); ( e ) a rat plasma sample collected 30 min after i.g. administration of salidroside (100 mg/kg) with IS (200 ng·mL −1 ); ( f ) a rat plasma sample collected 2 h after i.g. administration of salidroside (100 mg/kg) with IS (200 ng·mL −1 ); ( g ) a rat plasma sample collected 4 h after i.g. administration of salidroside (100 mg/kg) with IS (200 ng·mL −1 ).

Techniques Used:

Product ion mass spectra of [M−H] − . ( a ) Salidroside ([M−H] − , m/z 299.0); ( b ) p- Tyrosol ([M−H] − , m/z 137.0); ( c ) IS (paracetamol) ([M−H] − , m/z 150.1).
Figure Legend Snippet: Product ion mass spectra of [M−H] − . ( a ) Salidroside ([M−H] − , m/z 299.0); ( b ) p- Tyrosol ([M−H] − , m/z 137.0); ( c ) IS (paracetamol) ([M−H] − , m/z 150.1).

Techniques Used:

Mean concentration-time profiles in rat plasma (n = 6) obtained after i.v. administration of salidroside (i.v. 50 mg/kg) and i.g. administration of salidroside (i.g. 100 mg/kg). ( a : salidroside; b : p- tyrosol).
Figure Legend Snippet: Mean concentration-time profiles in rat plasma (n = 6) obtained after i.v. administration of salidroside (i.v. 50 mg/kg) and i.g. administration of salidroside (i.g. 100 mg/kg). ( a : salidroside; b : p- tyrosol).

Techniques Used: Concentration Assay

Representative chromatograms of: ( a ) a blank rat plasma sample; ( b ) a blank rat plasma spiked with salidroside and p- tyrosol (20 μg/mL); ( c ) a rat plasma sample collected 5 min after i.v. administration of salidroside (50 mg/kg); ( d ) a rat plasma sample collected 30 min after i.g. administration of salidroside (100 mg/kg). (1: salidroside, 2: p -tyrosol).
Figure Legend Snippet: Representative chromatograms of: ( a ) a blank rat plasma sample; ( b ) a blank rat plasma spiked with salidroside and p- tyrosol (20 μg/mL); ( c ) a rat plasma sample collected 5 min after i.v. administration of salidroside (50 mg/kg); ( d ) a rat plasma sample collected 30 min after i.g. administration of salidroside (100 mg/kg). (1: salidroside, 2: p -tyrosol).

Techniques Used:

Chemical structures of ( a ) salidroside; ( b ) p- tyrosol; and ( c ) paracetamol (IS).
Figure Legend Snippet: Chemical structures of ( a ) salidroside; ( b ) p- tyrosol; and ( c ) paracetamol (IS).

Techniques Used:

11) Product Images from "Plant Defensive β-Glucosidases Resist Digestion and Sustain Activity in the Gut of a Lepidopteran Herbivore"

Article Title: Plant Defensive β-Glucosidases Resist Digestion and Sustain Activity in the Gut of a Lepidopteran Herbivore

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2018.01389

Maize β-glucosidases were recovered in active form after digestion by S. littoralis larvae. Enzymatic activity levels were determined in non-ingested material and after digestion of diet cubes spiked with semi-purified maize β-glucosidase (A) , or after feeding on maize leaf tissue (B) . Activities were measured either using the non-specific β-glucosidase substrate pNPG (A) or the endogenous plant substrate DIMBOA-Glc (B) . Shown are the means ± standard errors [ N = 3 in both analyses; P = 0.2 (A) and P = 0.3 (B) ].
Figure Legend Snippet: Maize β-glucosidases were recovered in active form after digestion by S. littoralis larvae. Enzymatic activity levels were determined in non-ingested material and after digestion of diet cubes spiked with semi-purified maize β-glucosidase (A) , or after feeding on maize leaf tissue (B) . Activities were measured either using the non-specific β-glucosidase substrate pNPG (A) or the endogenous plant substrate DIMBOA-Glc (B) . Shown are the means ± standard errors [ N = 3 in both analyses; P = 0.2 (A) and P = 0.3 (B) ].

Techniques Used: Activity Assay, Purification, Gas Chromatography

Schematic representation of some reactions catalyzed by plant β-glucosidases. (A) Activation of glucosinolates by myrosinase. (B) Step-wise hydrolysis of the cyanogenic diglucoside amygdalin. (C) Hydrolytic activation of the benzoxazinoid glucoside DIMBOA-Glc and formation of the open-ring aglycone form in solution.
Figure Legend Snippet: Schematic representation of some reactions catalyzed by plant β-glucosidases. (A) Activation of glucosinolates by myrosinase. (B) Step-wise hydrolysis of the cyanogenic diglucoside amygdalin. (C) Hydrolytic activation of the benzoxazinoid glucoside DIMBOA-Glc and formation of the open-ring aglycone form in solution.

Techniques Used: Activation Assay, Gas Chromatography

12) Product Images from "The Metabolism of Salidroside to Its Aglycone p-Tyrosol in Rats following the Administration of Salidroside"

Article Title: The Metabolism of Salidroside to Its Aglycone p-Tyrosol in Rats following the Administration of Salidroside

Journal: PLoS ONE

doi: 10.1371/journal.pone.0103648

Mean concentration-time profiles of (A) salidroside and (B) p- tyrosol in rat tissues (n = 6) obtained after i.v. administration of salidroside (i.v. 50 mg/kg).
Figure Legend Snippet: Mean concentration-time profiles of (A) salidroside and (B) p- tyrosol in rat tissues (n = 6) obtained after i.v. administration of salidroside (i.v. 50 mg/kg).

Techniques Used: Concentration Assay

Mean concentration-time profiles of (A) salidroside and (B) p- tyrosol in rat tissues (n = 6) obtained after i.g. administration of salidroside (i.g. 100 mg/kg).
Figure Legend Snippet: Mean concentration-time profiles of (A) salidroside and (B) p- tyrosol in rat tissues (n = 6) obtained after i.g. administration of salidroside (i.g. 100 mg/kg).

Techniques Used: Concentration Assay

Chemical structures of (A) salidroside, (B) p- tyrosol and (C) paracetamol (IS).
Figure Legend Snippet: Chemical structures of (A) salidroside, (B) p- tyrosol and (C) paracetamol (IS).

Techniques Used:

MRM chromatograms of salidroside, p -tyrosol and the IS in (A) a blank rat liver tissue homogenate sample, (B) a blank rat liver tissue sample spiked with salidroside (500 ng/mL), p -tyrosol (500 ng/mL) and the IS (200 ng/mL), (C) a rat liver tissue homogenate sample collected 0.17 h after i.v. administration of salidroside (50 mg/kg) with the IS (200 ng/mL), (D) a rat liver tissue homogenate sample collected 1 h after i.g. administration of salidroside (100 mg/kg) with the IS (200 ng/mL).
Figure Legend Snippet: MRM chromatograms of salidroside, p -tyrosol and the IS in (A) a blank rat liver tissue homogenate sample, (B) a blank rat liver tissue sample spiked with salidroside (500 ng/mL), p -tyrosol (500 ng/mL) and the IS (200 ng/mL), (C) a rat liver tissue homogenate sample collected 0.17 h after i.v. administration of salidroside (50 mg/kg) with the IS (200 ng/mL), (D) a rat liver tissue homogenate sample collected 1 h after i.g. administration of salidroside (100 mg/kg) with the IS (200 ng/mL).

Techniques Used:

13) Product Images from "MASTL overexpression promotes chromosome instability and metastasis in breast cancer"

Article Title: MASTL overexpression promotes chromosome instability and metastasis in breast cancer

Journal: Oncogene

doi: 10.1038/s41388-018-0295-z

MASTL overexpression results in the loss of cell–cell junctions, causing migration defects. a Representative maximum projection images from confocal immunofluorescence of control and MASTL stained with H33342 (cyan) for E-Cadherin (green), β-catenin (yellow), and phalloidin (F-actin, pink). Scale bar 10 µm. b Wound-healing assays of control, and MASTL cell lines. Representative phase contrast, with inset of invading cell front (Control) and individual cells (MASTL) shown. Scale bar 50 µm. Kymographs (mCherry) generated by horizontal line through middle of wound area. Yellow dotted line indicates wound closure. c MRI Wound-Healing Tool for ImageJ was used to determine unclosed wound area as a function of time (mean ± SEM shown). Dotted line indicates 50% closure. Trend-line analysis performed using asymmetric sigmoidal analysis in Prism ( R 2 = 0.5888; Control and 0.7912; MASTL). d The MTrackJ plugin for ImageJ was used to track individual cells ( n = minimum 50 cell/condition) from b . e Data from d were analysed using the DiPer software tool. Shown are average speed and directionality ratios for MASTL and controls from three independent experiments (mean ± SEM, unpaired t -test, **** p
Figure Legend Snippet: MASTL overexpression results in the loss of cell–cell junctions, causing migration defects. a Representative maximum projection images from confocal immunofluorescence of control and MASTL stained with H33342 (cyan) for E-Cadherin (green), β-catenin (yellow), and phalloidin (F-actin, pink). Scale bar 10 µm. b Wound-healing assays of control, and MASTL cell lines. Representative phase contrast, with inset of invading cell front (Control) and individual cells (MASTL) shown. Scale bar 50 µm. Kymographs (mCherry) generated by horizontal line through middle of wound area. Yellow dotted line indicates wound closure. c MRI Wound-Healing Tool for ImageJ was used to determine unclosed wound area as a function of time (mean ± SEM shown). Dotted line indicates 50% closure. Trend-line analysis performed using asymmetric sigmoidal analysis in Prism ( R 2 = 0.5888; Control and 0.7912; MASTL). d The MTrackJ plugin for ImageJ was used to track individual cells ( n = minimum 50 cell/condition) from b . e Data from d were analysed using the DiPer software tool. Shown are average speed and directionality ratios for MASTL and controls from three independent experiments (mean ± SEM, unpaired t -test, **** p

Techniques Used: Over Expression, Migration, Immunofluorescence, Staining, Generated, Magnetic Resonance Imaging, Software

14) Product Images from "Removal of Abnormal Myofilament O-GlcNAcylation Restores Ca2+ Sensitivity in Diabetic Cardiac Muscle"

Article Title: Removal of Abnormal Myofilament O-GlcNAcylation Restores Ca2+ Sensitivity in Diabetic Cardiac Muscle

Journal: Diabetes

doi: 10.2337/db14-1107

Differential OGT and OGA subcellular localization and myofilament interactions in control and diabetic myocardium. Representative transmission electron microscopy images of control ( A ) and STZ diabetic rat myocardium ( B ). Ultrathin sections were examined with immunoelectron microscopy with primary antibodies (anti-OGT AL-28 and anti-OGA), and secondary antibodies gold-labeled (anti-rabbit, 12 nm; anti-chicken, 6 nm). Z-line, green arrowhead; OGT, purple circles; OGA, red circles; OGT and OGA in close vicinity, pink circles; OGT, purple arrowhead; and OGA, red arrowhead. Quantification of OGT ( C ) and OGA ( D ) number of particles/field in nine fields shows an increase for OGT (2 ± 0.6 vs. 8.6 ± 2.6, * P ≤ 0.028) and OGA (9.4 ± 2.9 vs 37.6 ± 12.6, * P ≤ 0.05) immunoelectron microscopy in STZ diabetic myocardium. Images were analyzed in ImageJ software (NIH). Representative coimmunoprecipitations (co-IP) of OGT ( E ) and OGA ( F ), followed by Western blots (WB) for α-sarcomeric actin, Tm, and MLC 1. A fraction of the inputs from controls (C1, C2), STZ (S1, S2), and agarose beads with no antibody (M) or isotype-specific normal antibody + agarose beads (1°) were included. G : Analysis of integrated signal density of myofilament immunoreactivity normalized to total IP OGT displayed a trend toward increased interactions with Tm and MLC 1. H : OGA co-IP shows that diabetic STZ rats display several fold increased associations with α-actin, α-Tm, and MLC 1. * P ≤ 0.05.
Figure Legend Snippet: Differential OGT and OGA subcellular localization and myofilament interactions in control and diabetic myocardium. Representative transmission electron microscopy images of control ( A ) and STZ diabetic rat myocardium ( B ). Ultrathin sections were examined with immunoelectron microscopy with primary antibodies (anti-OGT AL-28 and anti-OGA), and secondary antibodies gold-labeled (anti-rabbit, 12 nm; anti-chicken, 6 nm). Z-line, green arrowhead; OGT, purple circles; OGA, red circles; OGT and OGA in close vicinity, pink circles; OGT, purple arrowhead; and OGA, red arrowhead. Quantification of OGT ( C ) and OGA ( D ) number of particles/field in nine fields shows an increase for OGT (2 ± 0.6 vs. 8.6 ± 2.6, * P ≤ 0.028) and OGA (9.4 ± 2.9 vs 37.6 ± 12.6, * P ≤ 0.05) immunoelectron microscopy in STZ diabetic myocardium. Images were analyzed in ImageJ software (NIH). Representative coimmunoprecipitations (co-IP) of OGT ( E ) and OGA ( F ), followed by Western blots (WB) for α-sarcomeric actin, Tm, and MLC 1. A fraction of the inputs from controls (C1, C2), STZ (S1, S2), and agarose beads with no antibody (M) or isotype-specific normal antibody + agarose beads (1°) were included. G : Analysis of integrated signal density of myofilament immunoreactivity normalized to total IP OGT displayed a trend toward increased interactions with Tm and MLC 1. H : OGA co-IP shows that diabetic STZ rats display several fold increased associations with α-actin, α-Tm, and MLC 1. * P ≤ 0.05.

Techniques Used: Transmission Assay, Electron Microscopy, Immuno-Electron Microscopy, Labeling, Software, Co-Immunoprecipitation Assay, Western Blot

15) Product Images from "ELTA: Enzymatic Labeling of Terminal ADP-ribose"

Article Title: ELTA: Enzymatic Labeling of Terminal ADP-ribose

Journal: Molecular cell

doi: 10.1016/j.molcel.2018.12.022

ELTA labels free or protein-conjugated ADP-ribose monomers and polymers. (a) Schematics of ELTA. Free or protein-conjugated ADP-ribose can be labeled by incubating with OAS1 and dATP, where the 2’-OH terminus is indicated in red. Colored box indicates various dATP analogs that can also be used in the ELTA reactions, including radioactive ( 32 P), fluorescent (Cy3, Cy5), biotinylated or clickable analogs. (b) 15% urea-PAGE analyses of the addition of 32 P-dAMP onto ADP-ribose monomers and polymers using ELTA and visualized by autoradiograph. (c) MALDI-TOF analyses of the reaction of ADP-ribose with dATP, and with or without OAS1. (d-e) Analyses of the ELTA labeling reaction of (d) MARylated PARP10 catalytic domain (mod-PARP10 cd ) and (e) PARylated ha PARP (mod- ha PARP) using 32 P-dATP. Shown are a coomassie gel (left), an autoradiograph (middle), and a western blot probed with pan-ADP-ribose reagent (right). As negative controls, modified proteins were treated with the phosphodiesterase hs NudT16 to remove the 2’-OH termini of the ADP-ribose groups prior to ELTA labeling. For panel d, * indicates PARP10; OAS1 was ADP-ribosylated by PARP10 with the remnant of NAD + , and, therefore, detected by pan-ADP-ribose reagent and labeled by OAS1. For panel e, * indicates ha PARP and § indicates ha PARP fragments that were also ADP-ribosylated and, therefore, detected by pan-ADP-ribose reagent and labeled by OAS1.
Figure Legend Snippet: ELTA labels free or protein-conjugated ADP-ribose monomers and polymers. (a) Schematics of ELTA. Free or protein-conjugated ADP-ribose can be labeled by incubating with OAS1 and dATP, where the 2’-OH terminus is indicated in red. Colored box indicates various dATP analogs that can also be used in the ELTA reactions, including radioactive ( 32 P), fluorescent (Cy3, Cy5), biotinylated or clickable analogs. (b) 15% urea-PAGE analyses of the addition of 32 P-dAMP onto ADP-ribose monomers and polymers using ELTA and visualized by autoradiograph. (c) MALDI-TOF analyses of the reaction of ADP-ribose with dATP, and with or without OAS1. (d-e) Analyses of the ELTA labeling reaction of (d) MARylated PARP10 catalytic domain (mod-PARP10 cd ) and (e) PARylated ha PARP (mod- ha PARP) using 32 P-dATP. Shown are a coomassie gel (left), an autoradiograph (middle), and a western blot probed with pan-ADP-ribose reagent (right). As negative controls, modified proteins were treated with the phosphodiesterase hs NudT16 to remove the 2’-OH termini of the ADP-ribose groups prior to ELTA labeling. For panel d, * indicates PARP10; OAS1 was ADP-ribosylated by PARP10 with the remnant of NAD + , and, therefore, detected by pan-ADP-ribose reagent and labeled by OAS1. For panel e, * indicates ha PARP and § indicates ha PARP fragments that were also ADP-ribosylated and, therefore, detected by pan-ADP-ribose reagent and labeled by OAS1.

Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Western Blot, Modification

16) Product Images from "ELTA: Enzymatic Labeling of Terminal ADP-ribose"

Article Title: ELTA: Enzymatic Labeling of Terminal ADP-ribose

Journal: Molecular cell

doi: 10.1016/j.molcel.2018.12.022

ELTA labels free or protein-conjugated ADP-ribose monomers and polymers. (a) Schematics of ELTA. Free or protein-conjugated ADP-ribose can be labeled by incubating with OAS1 and dATP, where the 2’-OH terminus is indicated in red. Colored box indicates various dATP analogs that can also be used in the ELTA reactions, including radioactive ( 32 P), fluorescent (Cy3, Cy5), biotinylated or clickable analogs. (b) 15% urea-PAGE analyses of the addition of 32 P-dAMP onto ADP-ribose monomers and polymers using ELTA and visualized by autoradiograph. (c) MALDI-TOF analyses of the reaction of ADP-ribose with dATP, and with or without OAS1. (d-e) Analyses of the ELTA labeling reaction of (d) MARylated PARP10 catalytic domain (mod-PARP10 cd ) and (e) PARylated ha PARP (mod- ha PARP) using 32 P-dATP. Shown are a coomassie gel (left), an autoradiograph (middle), and a western blot probed with pan-ADP-ribose reagent (right). As negative controls, modified proteins were treated with the phosphodiesterase hs NudT16 to remove the 2’-OH termini of the ADP-ribose groups prior to ELTA labeling. For panel d, * indicates PARP10; OAS1 was ADP-ribosylated by PARP10 with the remnant of NAD + , and, therefore, detected by pan-ADP-ribose reagent and labeled by OAS1. For panel e, * indicates ha PARP and § indicates ha PARP fragments that were also ADP-ribosylated and, therefore, detected by pan-ADP-ribose reagent and labeled by OAS1.
Figure Legend Snippet: ELTA labels free or protein-conjugated ADP-ribose monomers and polymers. (a) Schematics of ELTA. Free or protein-conjugated ADP-ribose can be labeled by incubating with OAS1 and dATP, where the 2’-OH terminus is indicated in red. Colored box indicates various dATP analogs that can also be used in the ELTA reactions, including radioactive ( 32 P), fluorescent (Cy3, Cy5), biotinylated or clickable analogs. (b) 15% urea-PAGE analyses of the addition of 32 P-dAMP onto ADP-ribose monomers and polymers using ELTA and visualized by autoradiograph. (c) MALDI-TOF analyses of the reaction of ADP-ribose with dATP, and with or without OAS1. (d-e) Analyses of the ELTA labeling reaction of (d) MARylated PARP10 catalytic domain (mod-PARP10 cd ) and (e) PARylated ha PARP (mod- ha PARP) using 32 P-dATP. Shown are a coomassie gel (left), an autoradiograph (middle), and a western blot probed with pan-ADP-ribose reagent (right). As negative controls, modified proteins were treated with the phosphodiesterase hs NudT16 to remove the 2’-OH termini of the ADP-ribose groups prior to ELTA labeling. For panel d, * indicates PARP10; OAS1 was ADP-ribosylated by PARP10 with the remnant of NAD + , and, therefore, detected by pan-ADP-ribose reagent and labeled by OAS1. For panel e, * indicates ha PARP and § indicates ha PARP fragments that were also ADP-ribosylated and, therefore, detected by pan-ADP-ribose reagent and labeled by OAS1.

Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Western Blot, Modification

Detection of ADP-ribose length from individual proteins and cells using ELTA. (a) 15% urea-PAGE analyses of the ELTA labeling reaction of ADP-ribose monomer (lane 1) and PAR of mixed length (lane 2), as well as ADP-ribose monomers and polymers isolated from PARP1 automodification reactions with 1 mM NAD + for 0 (lane 3), 10 (lane 4), or 30 min (lane 5) that were labeled by OAS1 and 32 P-dATP. As a comparison, the ADP-ribose isolated from PARP1 automodification reaction in the same time frame with 1 mM NAD + with a trace of 32 P-NAD + were loaded in lanes 6–8. We note that 50-fold less of the reaction were loaded in lanes 3–5 compared with lanes 6–8. (b) 15% urea-PAGE analyses of ELTA labeling reaction of ADP-ribose monomers and polymers isolated from automodification of PARP1 along with either BSA and HPF1. The first lane contained ELTA-labeling of an equal mole of 5-, 10- and 20-mer PAR. The reaction in the PARP1+BSA lane was diluted 15 times in water prior to ELTA labeling. (c) 15% urea-PAGE analyses of the ELTA labeling reaction of ADP-ribose isolated from in vitro modified ha PARP (lane 1), from untreated HaCaT cells (lane 2), from HaCaT cells treated with 1 mM H 2 O 2 for 10 min (lane 3), from HaCaT cells treated with 1 mM H 2 O 2 for 10 min, but pre-treated the cells with 20 μM PARP inhibitor Olaparib for 2 h (lane 4), or pre-treated with 1 μM PARG inhibitor PDD00017273 for 2 h (lane 5). Corresponding lysates of cells from lanes 2–5 were probed with β-actin.
Figure Legend Snippet: Detection of ADP-ribose length from individual proteins and cells using ELTA. (a) 15% urea-PAGE analyses of the ELTA labeling reaction of ADP-ribose monomer (lane 1) and PAR of mixed length (lane 2), as well as ADP-ribose monomers and polymers isolated from PARP1 automodification reactions with 1 mM NAD + for 0 (lane 3), 10 (lane 4), or 30 min (lane 5) that were labeled by OAS1 and 32 P-dATP. As a comparison, the ADP-ribose isolated from PARP1 automodification reaction in the same time frame with 1 mM NAD + with a trace of 32 P-NAD + were loaded in lanes 6–8. We note that 50-fold less of the reaction were loaded in lanes 3–5 compared with lanes 6–8. (b) 15% urea-PAGE analyses of ELTA labeling reaction of ADP-ribose monomers and polymers isolated from automodification of PARP1 along with either BSA and HPF1. The first lane contained ELTA-labeling of an equal mole of 5-, 10- and 20-mer PAR. The reaction in the PARP1+BSA lane was diluted 15 times in water prior to ELTA labeling. (c) 15% urea-PAGE analyses of the ELTA labeling reaction of ADP-ribose isolated from in vitro modified ha PARP (lane 1), from untreated HaCaT cells (lane 2), from HaCaT cells treated with 1 mM H 2 O 2 for 10 min (lane 3), from HaCaT cells treated with 1 mM H 2 O 2 for 10 min, but pre-treated the cells with 20 μM PARP inhibitor Olaparib for 2 h (lane 4), or pre-treated with 1 μM PARG inhibitor PDD00017273 for 2 h (lane 5). Corresponding lysates of cells from lanes 2–5 were probed with β-actin.

Techniques Used: Polyacrylamide Gel Electrophoresis, Labeling, Isolation, In Vitro, Modification

17) Product Images from "CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation *"

Article Title: CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.017483

CAPS is phosphorylated at N- and C-terminal Ser residues. A , MALDI-TOF spectra of CAPS phosphopeptides. Phosphopeptides enriched from CAPS tryptic peptides by Ga(III)- immobilized metal ion affinity chromatography were incubated without ( upper panel )
Figure Legend Snippet: CAPS is phosphorylated at N- and C-terminal Ser residues. A , MALDI-TOF spectra of CAPS phosphopeptides. Phosphopeptides enriched from CAPS tryptic peptides by Ga(III)- immobilized metal ion affinity chromatography were incubated without ( upper panel )

Techniques Used: Affinity Chromatography, Incubation

18) Product Images from "Identification of Clostridium spp. derived from a sheep and cattle slaughterhouse by matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rDNA sequencing"

Article Title: Identification of Clostridium spp. derived from a sheep and cattle slaughterhouse by matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rDNA sequencing

Journal: Journal of Food Science and Technology

doi: 10.1007/s13197-018-3255-2

Comparing direct transfer and extended direct transfer sample preparation methods for identification of Clostridium species by MALDI-TOF MS at different days of anaerobic incubation (1, 3, and 5). Each isolate was tested three times for each method of sample preparation at each day
Figure Legend Snippet: Comparing direct transfer and extended direct transfer sample preparation methods for identification of Clostridium species by MALDI-TOF MS at different days of anaerobic incubation (1, 3, and 5). Each isolate was tested three times for each method of sample preparation at each day

Techniques Used: Sample Prep, Mass Spectrometry, Incubation

Overview of Bruker MALDI-TOF MS identification of Clostridium isolates at the species level. MALDI-TOF MS identified 94% isolates while 6% of isolates were not identified by MALDI-TOF MS. These isolates were identified via 16S rDNA as C. estertheticum (n = 8), C. frigidicarnis (n = 5), and C. gasigenes (n = 3) species
Figure Legend Snippet: Overview of Bruker MALDI-TOF MS identification of Clostridium isolates at the species level. MALDI-TOF MS identified 94% isolates while 6% of isolates were not identified by MALDI-TOF MS. These isolates were identified via 16S rDNA as C. estertheticum (n = 8), C. frigidicarnis (n = 5), and C. gasigenes (n = 3) species

Techniques Used: Mass Spectrometry

19) Product Images from "Simultaneous untargeted and targeted metabolomics profiling of underivatized primary metabolites in sulfur-deficient barley by ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry"

Article Title: Simultaneous untargeted and targeted metabolomics profiling of underivatized primary metabolites in sulfur-deficient barley by ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry

Journal: Plant Methods

doi: 10.1186/s13007-018-0329-0

Extracted ion chromatogram of amino acids and sulfur metabolites—waters acquity UPLC HSS T3 column—ESI+: 1, Proline; 2, Isoleucine; 3, Leucine; 4, Asparagine; 5, Glutamine; 6, Lysine; 7, O-Acetyl-serine; 8, Methionine; 9, Histidine; 10, Phenylalanine; 11, Arginine; 12, Tyrosine; 13, Tryptophan; 14, Thiamine; 15, Glutathione reduced; 16, S-adenosyl-methionine; 17, Glutathione oxidized
Figure Legend Snippet: Extracted ion chromatogram of amino acids and sulfur metabolites—waters acquity UPLC HSS T3 column—ESI+: 1, Proline; 2, Isoleucine; 3, Leucine; 4, Asparagine; 5, Glutamine; 6, Lysine; 7, O-Acetyl-serine; 8, Methionine; 9, Histidine; 10, Phenylalanine; 11, Arginine; 12, Tyrosine; 13, Tryptophan; 14, Thiamine; 15, Glutathione reduced; 16, S-adenosyl-methionine; 17, Glutathione oxidized

Techniques Used:

20) Product Images from "A subcortical inhibitory signal for behavioral arrest in the thalamus"

Article Title: A subcortical inhibitory signal for behavioral arrest in the thalamus

Journal: Nature neuroscience

doi: 10.1038/nn.3951

Activation of glycinergic afferents interrupts ongoing cortical activity. a) Representative standardized frontal cortical LFP traces before (1) and during (2) the optogenetic activation of GlyT2 fibers in the IL. b) Wavelet spectrum of the cortical LFP showing the 33 s long activation period together with pre- and post-illumination period. Grey bars indicate the position of LFP samples in (a). Warm colors indicate higher power. c) Power spectra of the cortical LFPs in the 30 s preceding the stimulation (orange) and during photoactivation (blue) of the GlyT2 fibers in IL. d) Statistical comparison of the power spectra of the stimulated and control periods in one representative animal (n=25 stimulations). Gray bar indicates the frequency range which displayed statistically significant difference (2.14 Hz - 5.8 Hz, Mann-Whitney U test). In this range the highest p value was 0.00226 at 5.8 Hz (W=457). All other p values were lower. Error bars represent the s.e.m. au arbitrary unit.
Figure Legend Snippet: Activation of glycinergic afferents interrupts ongoing cortical activity. a) Representative standardized frontal cortical LFP traces before (1) and during (2) the optogenetic activation of GlyT2 fibers in the IL. b) Wavelet spectrum of the cortical LFP showing the 33 s long activation period together with pre- and post-illumination period. Grey bars indicate the position of LFP samples in (a). Warm colors indicate higher power. c) Power spectra of the cortical LFPs in the 30 s preceding the stimulation (orange) and during photoactivation (blue) of the GlyT2 fibers in IL. d) Statistical comparison of the power spectra of the stimulated and control periods in one representative animal (n=25 stimulations). Gray bar indicates the frequency range which displayed statistically significant difference (2.14 Hz - 5.8 Hz, Mann-Whitney U test). In this range the highest p value was 0.00226 at 5.8 Hz (W=457). All other p values were lower. Error bars represent the s.e.m. au arbitrary unit.

Techniques Used: Activation Assay, Activity Assay, MANN-WHITNEY

Glycinergic terminals in IL are multisynaptic, co-express GABA and display variable postsynaptic receptor composition. a) 3D reconstruction of a GlyT2::eGFP -positive terminal in IL from serial electron microscopic (EM) images, three of which are shown on the right. Green, synapses; magenta, puncta adherentia; dark blue, membrane of the terminal; light blue, glia; arrows, synapse; arrowheads puncta adherantia. b) Consecutive electron micrographs of a GlyT2:eGFP bouton (b) in the mouse IL immunostained for eGFP using preembedding silver staining (left), and for GABA using postembedding immunogold labeling (right). c) Comparison of random dendritic diameters (white bars) in the IL and the diameter of targets postsynaptic to GlyT2::eGFP terminals (black bars). Random dendrite diameters are also shown and as the ratio of summated perimeter of the dendrites in each bin (black line with diamonds), which better reflect the available membrane surfaces. d) Correlation between the synapse numbers of the GlyT2::eGFP boutons and the diameter of the postsynaptic IL dendrites. e) The average number of synapses with increasing distances from a given synapse in eGFP boutons in the IL. f) Electron micrograph of a GlyT2-immunopositive axon terminal in the human IL. green arrowheads, synapses g) White arrows point to colocalization of the γ2 subunit of GABA A receptors and of glycine receptors postsynaptic to GlyT2::eGFP terminals. The cityscape plot (h) represents the number of apposed GABAγ2R receptor and GlyR clusters per GlyT2::eGFP varicosity. Scales: a, b, f, 500 nm; g 1μm.
Figure Legend Snippet: Glycinergic terminals in IL are multisynaptic, co-express GABA and display variable postsynaptic receptor composition. a) 3D reconstruction of a GlyT2::eGFP -positive terminal in IL from serial electron microscopic (EM) images, three of which are shown on the right. Green, synapses; magenta, puncta adherentia; dark blue, membrane of the terminal; light blue, glia; arrows, synapse; arrowheads puncta adherantia. b) Consecutive electron micrographs of a GlyT2:eGFP bouton (b) in the mouse IL immunostained for eGFP using preembedding silver staining (left), and for GABA using postembedding immunogold labeling (right). c) Comparison of random dendritic diameters (white bars) in the IL and the diameter of targets postsynaptic to GlyT2::eGFP terminals (black bars). Random dendrite diameters are also shown and as the ratio of summated perimeter of the dendrites in each bin (black line with diamonds), which better reflect the available membrane surfaces. d) Correlation between the synapse numbers of the GlyT2::eGFP boutons and the diameter of the postsynaptic IL dendrites. e) The average number of synapses with increasing distances from a given synapse in eGFP boutons in the IL. f) Electron micrograph of a GlyT2-immunopositive axon terminal in the human IL. green arrowheads, synapses g) White arrows point to colocalization of the γ2 subunit of GABA A receptors and of glycine receptors postsynaptic to GlyT2::eGFP terminals. The cityscape plot (h) represents the number of apposed GABAγ2R receptor and GlyR clusters per GlyT2::eGFP varicosity. Scales: a, b, f, 500 nm; g 1μm.

Techniques Used: Silver Staining, Labeling

Glycinergic input evokes non-depressing inhibition and reduces IL cell firing. a) Scheme of the experiment. b) ChR2-eYFP containing fibers in the IL. c) Averaged sample trace of light-evoked IPSCs before (black trace) and after (red) application of gabazine and strychnine. d) Variable mixed GABA/glycinergic phenotype of light-evoked IPSCs. Three different examples are shown with only GABAergic (top), mixed GABA/glycinergic (center), and only glycinergic (bottom) transmission. e) Ratio of IL cells showing various proportions of glycinergic leIPSCs. f-g ) SR95531 application leads to a significant acceleration of the decay time course of the leIPSCs. See the averaged traces for a single recorded cell in (f), and the pooled results for all the experiments in (g). h) Light-evoked responses display little depression during stimulation trains at different frequencies. i) Activation of GlyT2 fibers interrupts firing of IL neurons recorded in the current clamp configuration.
Figure Legend Snippet: Glycinergic input evokes non-depressing inhibition and reduces IL cell firing. a) Scheme of the experiment. b) ChR2-eYFP containing fibers in the IL. c) Averaged sample trace of light-evoked IPSCs before (black trace) and after (red) application of gabazine and strychnine. d) Variable mixed GABA/glycinergic phenotype of light-evoked IPSCs. Three different examples are shown with only GABAergic (top), mixed GABA/glycinergic (center), and only glycinergic (bottom) transmission. e) Ratio of IL cells showing various proportions of glycinergic leIPSCs. f-g ) SR95531 application leads to a significant acceleration of the decay time course of the leIPSCs. See the averaged traces for a single recorded cell in (f), and the pooled results for all the experiments in (g). h) Light-evoked responses display little depression during stimulation trains at different frequencies. i) Activation of GlyT2 fibers interrupts firing of IL neurons recorded in the current clamp configuration.

Techniques Used: Inhibition, Transmission Assay, Activation Assay

Activation of glycinergic afferents interrupts ongoing behavior activity. a) Experimental design. b) Mice trajectory during the 1 st (red) and 2 nd (green) 5 s of optogenetic activation of GlyT2 fibers in the IL and during laser light shut off (black lines), in control (eYFP, left) and experimental (ChR2-eYFP, right) conditions. c) Average movement of control (dashed trace) and optogenetically activated (continuous trace) mice before, during (blue bar) and after stimulation. Error bars represent the s.e.m.
Figure Legend Snippet: Activation of glycinergic afferents interrupts ongoing behavior activity. a) Experimental design. b) Mice trajectory during the 1 st (red) and 2 nd (green) 5 s of optogenetic activation of GlyT2 fibers in the IL and during laser light shut off (black lines), in control (eYFP, left) and experimental (ChR2-eYFP, right) conditions. c) Average movement of control (dashed trace) and optogenetically activated (continuous trace) mice before, during (blue bar) and after stimulation. Error bars represent the s.e.m.

Techniques Used: Activation Assay, Activity Assay, Mouse Assay

Activity of GlyT2-positive neurons in the PRF in vivo is linked to cortical slow oscillation. a) Experimental design. b) Spiking activity of a GlyT2 cell in vivo under ketamine-xylazine anesthesia (bottom trace) together with the cortical LFP (top trace) and filtered cortical multiunit activity (MUA, middle trace). c) The recorded and neurobiotin filled cell display GlyT2::eGFP expression. d) Neurolucida reconstruction of the recorded cell . e-f) Phase distribution of the firing activity of five different GlyT2::eGFP -positive neurons relative to the cortical slow oscillation. One cycle is 360 o , 0 o peak of the UP state. Note the different phase preference of each cell. Scale: c, 20 µm; d, 100 µm.
Figure Legend Snippet: Activity of GlyT2-positive neurons in the PRF in vivo is linked to cortical slow oscillation. a) Experimental design. b) Spiking activity of a GlyT2 cell in vivo under ketamine-xylazine anesthesia (bottom trace) together with the cortical LFP (top trace) and filtered cortical multiunit activity (MUA, middle trace). c) The recorded and neurobiotin filled cell display GlyT2::eGFP expression. d) Neurolucida reconstruction of the recorded cell . e-f) Phase distribution of the firing activity of five different GlyT2::eGFP -positive neurons relative to the cortical slow oscillation. One cycle is 360 o , 0 o peak of the UP state. Note the different phase preference of each cell. Scale: c, 20 µm; d, 100 µm.

Techniques Used: Activity Assay, In Vivo, Expressing

Glycinergic afferents in the mouse and human IL. a) Injection site of the retrograde tracer fluorogold (FG) into the IL of a GlyT2::eGFP mouse. b-d) Retrogradely labeled GlyT2::eGFP -positive (arrows) and negative (arrowheads) neurons in the nucleus pontis oralis (PnO) at the coronal level indicated in the inset. e) Injection site of the anterograde tracer PHAL into the PnO and ( f-g) anterogradely labeled GlyT2::eGFP -positive fibers (arrows) in the IL at the position shown in the inset. Distribution of GlyT2 fibers in the mouse ( i,j) and human ( m,n) thalamus at two coronal levels. The figures represent cumulative data. Light microscopic images of GlyT2-positive fibers and innervation of calbindin-positive cells via multiple contacts in the mouse (k, l) and human ( o, p) IL. Scale bars: A,E, 1mm; all other 20 µm. CB, calbindin, For other abbreviations see Supplementary Fig 1,2 .
Figure Legend Snippet: Glycinergic afferents in the mouse and human IL. a) Injection site of the retrograde tracer fluorogold (FG) into the IL of a GlyT2::eGFP mouse. b-d) Retrogradely labeled GlyT2::eGFP -positive (arrows) and negative (arrowheads) neurons in the nucleus pontis oralis (PnO) at the coronal level indicated in the inset. e) Injection site of the anterograde tracer PHAL into the PnO and ( f-g) anterogradely labeled GlyT2::eGFP -positive fibers (arrows) in the IL at the position shown in the inset. Distribution of GlyT2 fibers in the mouse ( i,j) and human ( m,n) thalamus at two coronal levels. The figures represent cumulative data. Light microscopic images of GlyT2-positive fibers and innervation of calbindin-positive cells via multiple contacts in the mouse (k, l) and human ( o, p) IL. Scale bars: A,E, 1mm; all other 20 µm. CB, calbindin, For other abbreviations see Supplementary Fig 1,2 .

Techniques Used: Injection, Labeling

21) Product Images from "Cysteine residues contribute to the dimerization and enzymatic activity of human nuclear dUTP nucleotidohydrolase (nDut)"

Article Title: Cysteine residues contribute to the dimerization and enzymatic activity of human nuclear dUTP nucleotidohydrolase (nDut)

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1002/pro.3481

Cysteine 3 is a critical residue in stabilizing the secondary structure of nuclear dUTPase. The expression of nDut.Ctag, nDut.Ntag and cysteine to alanine mutants in U‐2 OS cells implicate C3 as a critical residue in stabilizing the higher order structure of n.Dut. Two constructs of each set of proteins contained either a hexahistidine C‐terminus tag (A, C) or hexahistidine N‐terminus tag (B, D). 3 μg of each plasmid were transiently transfected into U2‐OS cells followed by a 24‐h incubation. The cells were harvested and 20 μg of total cell extract was applied to a 4–20% tris‐glycine SDS‐PAGE (±) BME as indicated. Western blot analysis was then preformed using a histidine primary antibody. The predicted molecular weight of nDut.Ntag is 21,188 Da. This includes the existence of a 28 amino acid leader sequence (3458 Da). The nDut.Ctag construct is void of this leader sequence and is predicted to be 18,571 Da. The lower molecular weight band observed in (A) and (C) is likely a truncated form of dUTPase (M24) (Fig. S3 ).
Figure Legend Snippet: Cysteine 3 is a critical residue in stabilizing the secondary structure of nuclear dUTPase. The expression of nDut.Ctag, nDut.Ntag and cysteine to alanine mutants in U‐2 OS cells implicate C3 as a critical residue in stabilizing the higher order structure of n.Dut. Two constructs of each set of proteins contained either a hexahistidine C‐terminus tag (A, C) or hexahistidine N‐terminus tag (B, D). 3 μg of each plasmid were transiently transfected into U2‐OS cells followed by a 24‐h incubation. The cells were harvested and 20 μg of total cell extract was applied to a 4–20% tris‐glycine SDS‐PAGE (±) BME as indicated. Western blot analysis was then preformed using a histidine primary antibody. The predicted molecular weight of nDut.Ntag is 21,188 Da. This includes the existence of a 28 amino acid leader sequence (3458 Da). The nDut.Ctag construct is void of this leader sequence and is predicted to be 18,571 Da. The lower molecular weight band observed in (A) and (C) is likely a truncated form of dUTPase (M24) (Fig. S3 ).

Techniques Used: Expressing, Construct, Plasmid Preparation, Transfection, Incubation, SDS Page, Western Blot, Molecular Weight, Sequencing

An intermolecular disulfide bridge formation between two cysteine 3 residues is essential for nuclear dUTPase dimer formation. A 12% Tris‐Glycine SDS‐PAGE coomassie stained gel of recombinant Wt nDut.Ntag and cysteine to alanine mutants in (A) non‐reducing (‐BME) or (B) reducing conditions (+BME). (C) A 16% Tris‐Glycine SDS‐PAGE coomassie stained gel of recombinant Wt nDut.Ctag (+/‐) BME. Predicted molecular weights and migration behavior on SDS‐PAGE is reflective of what is seen in Figure 2 . Mass spectrometry was performed with 2 μg of both the full length Wt nDut.ntag (D) and the C3A mutant (E). The peak of interest corresponds to the dimeric (42 KDa) state of the protein which can be visualized in the Wt (D) spectrum. As shown, this peak is significantly diminished in the C3A spectrum.
Figure Legend Snippet: An intermolecular disulfide bridge formation between two cysteine 3 residues is essential for nuclear dUTPase dimer formation. A 12% Tris‐Glycine SDS‐PAGE coomassie stained gel of recombinant Wt nDut.Ntag and cysteine to alanine mutants in (A) non‐reducing (‐BME) or (B) reducing conditions (+BME). (C) A 16% Tris‐Glycine SDS‐PAGE coomassie stained gel of recombinant Wt nDut.Ctag (+/‐) BME. Predicted molecular weights and migration behavior on SDS‐PAGE is reflective of what is seen in Figure 2 . Mass spectrometry was performed with 2 μg of both the full length Wt nDut.ntag (D) and the C3A mutant (E). The peak of interest corresponds to the dimeric (42 KDa) state of the protein which can be visualized in the Wt (D) spectrum. As shown, this peak is significantly diminished in the C3A spectrum.

Techniques Used: SDS Page, Staining, Recombinant, Migration, Mass Spectrometry, Mutagenesis

22) Product Images from "UV laser-induced cross-linking in peptides"

Article Title: UV laser-induced cross-linking in peptides

Journal: Rapid communications in mass spectrometry : RCM

doi: 10.1002/rcm.6610

Positive-ion MALDI-TOF mass spectra of a mixture of xenopsin (M x ), interleukin (M i ) and angiotensin I (M a ) not irradiated (panel A) and irradiated for 10 sec (panel B).
Figure Legend Snippet: Positive-ion MALDI-TOF mass spectra of a mixture of xenopsin (M x ), interleukin (M i ) and angiotensin I (M a ) not irradiated (panel A) and irradiated for 10 sec (panel B).

Techniques Used: Irradiation, Size-exclusion Chromatography

23) Product Images from "Structure of the DEAH/RHA ATPase Prp43p bound to RNA implicates a pair of hairpins and motif Va in translocation along RNA"

Article Title: Structure of the DEAH/RHA ATPase Prp43p bound to RNA implicates a pair of hairpins and motif Va in translocation along RNA

Journal: RNA

doi: 10.1261/rna.060954.117

Motif Ib is not required for RNA binding or ATPase activity but is required for unwinding a 3′ overhang duplex. ( A ) Mutations in the tip of the 3′HP encompassing motif Ib (R177A or R177A/F178A) compromise unwinding of a 21-bp RNA/DNA duplex having a 27-nt 3′ RNA overhang. ( B ) Mutations in motif Ib permit RNA binding. Binding of Prp43 to the RNA unwinding substrate used in panel A was assayed by EMSA under conditions similar to the unwinding reactions but in the absence of ATP. The Prp43p–RNA complex is indicated. ( C ) Mutations in motif Ib permit ATPase activity. RNA-stimulated, Prp43-dependent ATPase activity was analyzed by TLC. The mutation E216A of motif II, the Walker B motif, was used as a negative control. ( D ) Unwinding of either a 3′ or 5′ unstructured 32-nt RNA overhang substrate with an identical 28-mer duplex. Heat denaturation of the duplex (Δ) was used as a positive control for unwinding. A line separating lanes 7 and 8 or lanes 15 and 16 indicates that a single gel image was cut and arranged to juxtapose relevant lanes.
Figure Legend Snippet: Motif Ib is not required for RNA binding or ATPase activity but is required for unwinding a 3′ overhang duplex. ( A ) Mutations in the tip of the 3′HP encompassing motif Ib (R177A or R177A/F178A) compromise unwinding of a 21-bp RNA/DNA duplex having a 27-nt 3′ RNA overhang. ( B ) Mutations in motif Ib permit RNA binding. Binding of Prp43 to the RNA unwinding substrate used in panel A was assayed by EMSA under conditions similar to the unwinding reactions but in the absence of ATP. The Prp43p–RNA complex is indicated. ( C ) Mutations in motif Ib permit ATPase activity. RNA-stimulated, Prp43-dependent ATPase activity was analyzed by TLC. The mutation E216A of motif II, the Walker B motif, was used as a negative control. ( D ) Unwinding of either a 3′ or 5′ unstructured 32-nt RNA overhang substrate with an identical 28-mer duplex. Heat denaturation of the duplex (Δ) was used as a positive control for unwinding. A line separating lanes 7 and 8 or lanes 15 and 16 indicates that a single gel image was cut and arranged to juxtapose relevant lanes.

Techniques Used: RNA Binding Assay, Activity Assay, Binding Assay, Thin Layer Chromatography, Mutagenesis, Negative Control, Positive Control

Structure of Prp43p bound to ADPNP and RNA
Figure Legend Snippet: Structure of Prp43p bound to ADPNP and RNA

Techniques Used:

The rotation of RecA2 enables RNA and ADPNP binding, and alternative motif Va conformations implicate this element as an ATP sensor. ( A , B ) The 9° rotation of RecA2 enables binding of both RNA ( A ) and ATP ( B ), implicating a mechanism by which RNA binding stimulates ATPase activity. In panel A , the movement of motifs IV and IVa in the RNA- and ADPNP-bound structure, relative to the ADP-bound structure (PDB 3KX2), is indicated by arrows. In panel B , the movement of motif VI in the RNA- and ADPNP-bound structure, relative to the ADP-bound structure, is indicated by arrows. In panels A and B , the RecA1 domain of Prp43p in the RNA-free, ADP-bound (gray) and the RNA- and ADPNP-bound (colored) states were superimposed. ( C , D ) In Prp43, as in NS3, motif Va rearranges, and movement correlates with loss of the γ-phosphate. Panel C compares the ADPNP- and RNA-bound state versus ADP-bound state of Prp43p; panel D compares the ADP–BeF 3 - and RNA-bound state versus the apo, RNA-bound state of NS3 (PDB 3O8R versus 3O8C). In each case, the RecA2 domain of the ATP-bound state (colored) was superimposed with the RecA2 domain of the contrasting states (gray). Blue highlights the static nature of motif V while red highlights the dynamic nature of motif Va. The ATP analog is shown, colored by atom type. RNA is omitted for clarity.
Figure Legend Snippet: The rotation of RecA2 enables RNA and ADPNP binding, and alternative motif Va conformations implicate this element as an ATP sensor. ( A , B ) The 9° rotation of RecA2 enables binding of both RNA ( A ) and ATP ( B ), implicating a mechanism by which RNA binding stimulates ATPase activity. In panel A , the movement of motifs IV and IVa in the RNA- and ADPNP-bound structure, relative to the ADP-bound structure (PDB 3KX2), is indicated by arrows. In panel B , the movement of motif VI in the RNA- and ADPNP-bound structure, relative to the ADP-bound structure, is indicated by arrows. In panels A and B , the RecA1 domain of Prp43p in the RNA-free, ADP-bound (gray) and the RNA- and ADPNP-bound (colored) states were superimposed. ( C , D ) In Prp43, as in NS3, motif Va rearranges, and movement correlates with loss of the γ-phosphate. Panel C compares the ADPNP- and RNA-bound state versus ADP-bound state of Prp43p; panel D compares the ADP–BeF 3 - and RNA-bound state versus the apo, RNA-bound state of NS3 (PDB 3O8R versus 3O8C). In each case, the RecA2 domain of the ATP-bound state (colored) was superimposed with the RecA2 domain of the contrasting states (gray). Blue highlights the static nature of motif V while red highlights the dynamic nature of motif Va. The ATP analog is shown, colored by atom type. RNA is omitted for clarity.

Techniques Used: Binding Assay, RNA Binding Assay, Activity Assay

24) Product Images from "Periplasmic production via the pET expression system of soluble, bioactive human growth hormone"

Article Title: Periplasmic production via the pET expression system of soluble, bioactive human growth hormone

Journal: Protein expression and purification

doi: 10.1016/j.pep.2012.11.002

Size exclusion chromatography (a–c) and MALF-TOF (d–f) analysis of TEV-TROPIN (a, d), pelB-hGH (b, e), and ompA-hGH (c, f) The retention time of the SEC calibration standards are indicated by tick marks on the x-axis. The standards include thyroglobulin 670 kDa, gamma-globulin 158 kDa, ovalbumin 44kDa, myoglobin 17 kDa, and vitamin B12 1.35 kDa. The unlabeled tick under the hGH peak in each panel corresponds to the retention time of the 17 kDa myoglobin standard. Obs: = Observed, Exp: = Expected.
Figure Legend Snippet: Size exclusion chromatography (a–c) and MALF-TOF (d–f) analysis of TEV-TROPIN (a, d), pelB-hGH (b, e), and ompA-hGH (c, f) The retention time of the SEC calibration standards are indicated by tick marks on the x-axis. The standards include thyroglobulin 670 kDa, gamma-globulin 158 kDa, ovalbumin 44kDa, myoglobin 17 kDa, and vitamin B12 1.35 kDa. The unlabeled tick under the hGH peak in each panel corresponds to the retention time of the 17 kDa myoglobin standard. Obs: = Observed, Exp: = Expected.

Techniques Used: Size-exclusion Chromatography

Reducing and non-reducing SDS-PAGE analysis of recombinant hGH Reduced TEV-TROPIN (lane 1), non-reduced TEV-TROPIN (lane 2), reduced pelB-hGH (lane 3), non-reduced pelB-hGH (lane 4), reduced ompA-hGH (lane 5), non-reduced ompA-hGH (lane 6). 7.5 μg of protein were loaded in each lane.
Figure Legend Snippet: Reducing and non-reducing SDS-PAGE analysis of recombinant hGH Reduced TEV-TROPIN (lane 1), non-reduced TEV-TROPIN (lane 2), reduced pelB-hGH (lane 3), non-reduced pelB-hGH (lane 4), reduced ompA-hGH (lane 5), non-reduced ompA-hGH (lane 6). 7.5 μg of protein were loaded in each lane.

Techniques Used: SDS Page, Recombinant

25) Product Images from "Interaction with phospholipids modulates ?-synuclein nitration and lipid-protein adduct formation"

Article Title: Interaction with phospholipids modulates ?-synuclein nitration and lipid-protein adduct formation

Journal: Biochemical Journal

doi: 10.1042/BJ20051277

Unsaturated fatty acids modulate peroxynitrite-mediated α-syn aggregation
Figure Legend Snippet: Unsaturated fatty acids modulate peroxynitrite-mediated α-syn aggregation

Techniques Used:

α-Syn binding to PC/PA liposomes modulates protein aggregation and nitration
Figure Legend Snippet: α-Syn binding to PC/PA liposomes modulates protein aggregation and nitration

Techniques Used: Binding Assay, Nitration

Formation of HNE–α-syn adducts
Figure Legend Snippet: Formation of HNE–α-syn adducts

Techniques Used:

Formation of lipid–protein adducts during peroxynitrite-mediated α-syn oxidation
Figure Legend Snippet: Formation of lipid–protein adducts during peroxynitrite-mediated α-syn oxidation

Techniques Used:

LC–MS and LC–MS/MS analyses of α-syn after tryptic digestion
Figure Legend Snippet: LC–MS and LC–MS/MS analyses of α-syn after tryptic digestion

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

α-Syn interacts with acidic phospholipids
Figure Legend Snippet: α-Syn interacts with acidic phospholipids

Techniques Used:

Lipid–protein adduct formation as an intermediate of α-syn aggregation
Figure Legend Snippet: Lipid–protein adduct formation as an intermediate of α-syn aggregation

Techniques Used:

26) Product Images from "A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation"

Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

Journal: Journal of Proteome Research

doi: 10.1021/acs.jproteome.8b00895

Protein–protein interactions (PPI) in IFN-γ-induced ADP-ribosylome on THP-1 cells. (A) A PPI network mapping all 145 ADPr proteins from Af1521 data and visualizing their interactions (confidence interaction scores ≥700). ADPr proteins with at least one interaction with another ADPr proteins are shown. Selected GO biological processes (GO-1 to GO-7 below) from the extended list in Table S4 . (B) PPI networks of 2-fold increased or decreased ADPr proteins in the IFN-γ compared to control. (C) PPI networks from a subset of 2-fold increasing or decreasing enriched proteins using the 10H workflow in response to IFN-γ stimulation.
Figure Legend Snippet: Protein–protein interactions (PPI) in IFN-γ-induced ADP-ribosylome on THP-1 cells. (A) A PPI network mapping all 145 ADPr proteins from Af1521 data and visualizing their interactions (confidence interaction scores ≥700). ADPr proteins with at least one interaction with another ADPr proteins are shown. Selected GO biological processes (GO-1 to GO-7 below) from the extended list in Table S4 . (B) PPI networks of 2-fold increased or decreased ADPr proteins in the IFN-γ compared to control. (C) PPI networks from a subset of 2-fold increasing or decreasing enriched proteins using the 10H workflow in response to IFN-γ stimulation.

Techniques Used:

ADP-ribosylation increases during IFN-γ-induced pro-inflammatory activation of macrophages. (A) Two independent strategies, Af1521 and 10H antibody workflows, for ADP-ribosylation proteomics. (B) Antipan ADP-ribose Western blot analysis of IFN-γ-treated THP-1 cells over 24 h. (C) IFN-γ activation replicates: two sets (control or IFN-γ) of macrophage activation were used for the Af1521 workflow, and three sets were used for the 10H antibody workflow. Details about ADPr peptide data acquisition and analysis are highlighted using the second Af1521 replicate of IFN-γ-treated THP-1 cells.
Figure Legend Snippet: ADP-ribosylation increases during IFN-γ-induced pro-inflammatory activation of macrophages. (A) Two independent strategies, Af1521 and 10H antibody workflows, for ADP-ribosylation proteomics. (B) Antipan ADP-ribose Western blot analysis of IFN-γ-treated THP-1 cells over 24 h. (C) IFN-γ activation replicates: two sets (control or IFN-γ) of macrophage activation were used for the Af1521 workflow, and three sets were used for the 10H antibody workflow. Details about ADPr peptide data acquisition and analysis are highlighted using the second Af1521 replicate of IFN-γ-treated THP-1 cells.

Techniques Used: Activation Assay, Western Blot

IFN-γ increased ARTD8/PARP14 and ARTD9/PARP9 ADP-ribosylation. (A) A pan ADP-ribose Western blot analysis of control and IFN-γ-treated THP-1 cells after 10H IP or incubation with IgG. (B) A comparison of enriched proteins using a Venn diagram between 10H and IgG. The plot showing the log 2 (abundance ratio (10H/IgG)) and −log( p -value) of 551 shared proteins (Venn diagram). 114 proteins passed the threshold of abundance ratio (10H/IgG) > 10-fold and p -value
Figure Legend Snippet: IFN-γ increased ARTD8/PARP14 and ARTD9/PARP9 ADP-ribosylation. (A) A pan ADP-ribose Western blot analysis of control and IFN-γ-treated THP-1 cells after 10H IP or incubation with IgG. (B) A comparison of enriched proteins using a Venn diagram between 10H and IgG. The plot showing the log 2 (abundance ratio (10H/IgG)) and −log( p -value) of 551 shared proteins (Venn diagram). 114 proteins passed the threshold of abundance ratio (10H/IgG) > 10-fold and p -value

Techniques Used: Western Blot, Incubation

27) Product Images from "The Oligomerization Domain of VP3, the Scaffolding Protein of Infectious Bursal Disease Virus, Plays a Critical Role in Capsid Assembly"

Article Title: The Oligomerization Domain of VP3, the Scaffolding Protein of Infectious Bursal Disease Virus, Plays a Critical Role in Capsid Assembly

Journal: Journal of Virology

doi: 10.1128/JVI.77.11.6438-6449.2003

Effect of VP1 coexpression on His-VP3 proteolytic cleavage. (A) Detection of VP3-VP1 complexes. H5 cells were infected with FB/His-VP3 or FB/His-VP3-VP1. At 72 h postinfection, cells were harvested, and the corresponding extracts were subjected to IMAC purification. Samples corresponding to total cell extracts (T) and IMAC-purified polypeptides (P) were analyzed by SDS-PAGE. After electrophoresis, gels were fixed and silver stained. The positions of molecular mass markers are indicated (in kilodaltons). (B) Western blot analysis of extracts from H5 cells infected with FB/His-VP3 or FB/His-VP3-VP1 or coinfected with FB/His-VP3 and FB/His-VP1. Infected cells were harvested at 72 h postinfection, resuspended in lysis buffer, and subjected to IMAC purification. Samples were subjected to SDS-PAGE and Western blot analysis with rabbit anti-VP3 serum, followed by addition of horseradish peroxidase-conjugated goat anti-rat immunoglobulin. The signal was detected by enhanced chemiluminescence. The positions of molecular mass markers (in kilodaltons) are indicated.
Figure Legend Snippet: Effect of VP1 coexpression on His-VP3 proteolytic cleavage. (A) Detection of VP3-VP1 complexes. H5 cells were infected with FB/His-VP3 or FB/His-VP3-VP1. At 72 h postinfection, cells were harvested, and the corresponding extracts were subjected to IMAC purification. Samples corresponding to total cell extracts (T) and IMAC-purified polypeptides (P) were analyzed by SDS-PAGE. After electrophoresis, gels were fixed and silver stained. The positions of molecular mass markers are indicated (in kilodaltons). (B) Western blot analysis of extracts from H5 cells infected with FB/His-VP3 or FB/His-VP3-VP1 or coinfected with FB/His-VP3 and FB/His-VP1. Infected cells were harvested at 72 h postinfection, resuspended in lysis buffer, and subjected to IMAC purification. Samples were subjected to SDS-PAGE and Western blot analysis with rabbit anti-VP3 serum, followed by addition of horseradish peroxidase-conjugated goat anti-rat immunoglobulin. The signal was detected by enhanced chemiluminescence. The positions of molecular mass markers (in kilodaltons) are indicated.

Techniques Used: Infection, Purification, SDS Page, Electrophoresis, Staining, Western Blot, Lysis

Mapping VP3 proteolytic cleavage site. (A) The diagram shows the set of VP3 C-terminal deletion mutants used for mapping the VP3 cleavage site. (B) Western blot analysis of IMAC-purified polypeptides encoded by the different VP3 C-terminal deletion mutants. Extracts from rBV-infected cells were subjected to IMAC. Affinity-purified polypeptides were subjected to SDS-PAGE and Western blot analysis with rabbit anti-VP3 serum, followed by addition of horseradish peroxidase-conjugated goat anti-rat immunoglobulin. The signal was detected by enhanced chemiluminescence. The positions of molecular mass markers are shown (in kilodaltons). Arrows indicate the position corresponding to full-length (F) and C-terminally trimmed (T) His-VP3 polypeptide products.
Figure Legend Snippet: Mapping VP3 proteolytic cleavage site. (A) The diagram shows the set of VP3 C-terminal deletion mutants used for mapping the VP3 cleavage site. (B) Western blot analysis of IMAC-purified polypeptides encoded by the different VP3 C-terminal deletion mutants. Extracts from rBV-infected cells were subjected to IMAC. Affinity-purified polypeptides were subjected to SDS-PAGE and Western blot analysis with rabbit anti-VP3 serum, followed by addition of horseradish peroxidase-conjugated goat anti-rat immunoglobulin. The signal was detected by enhanced chemiluminescence. The positions of molecular mass markers are shown (in kilodaltons). Arrows indicate the position corresponding to full-length (F) and C-terminally trimmed (T) His-VP3 polypeptide products.

Techniques Used: Western Blot, Purification, Infection, Affinity Purification, SDS Page

28) Product Images from "The S. pombe “cytokinesis” NDR kinase Sid2 activates Fin1 NIMA kinase to control mitotic commitment via Pom1/Wee1"

Article Title: The S. pombe “cytokinesis” NDR kinase Sid2 activates Fin1 NIMA kinase to control mitotic commitment via Pom1/Wee1

Journal: Nature cell biology

doi: 10.1038/ncb2514

Sid2 phosphorylation of Fin1 on serines 377, 526 and 698 promotes Fin1 activity in G2 phase before a peak of each kinase activity accompanies mitotic progression (a, b) Fin1 Kinase assays from size selected cultures in which His tagged Fin1.KD was labeled with 32 PγATP to quantitate activity that is plotted alongside the septation profile. (a) wild type (b) Small G2 sid2.as4 skp1.A4 cells were isolated from a culture grown at 25°C and immediately shifted to 36°C to inactivate Skp1 (and so preserve activated Fin1) at t=0. The culture was split in two and 20 μM 3-MB-PP1 (left assay) or solvent alone (right assay) added after the first division at t = 180. (c, d) Fin1 immunoprecipitates from asynchronous skp1.A4 (c) or cell size selected sid2.as4 skp1.A4 (d) cultures were split in two and probed with antibodies that recognise the indicated phosphorylation sites or polyclonal antibodies that recognise the non-catalytic domain of Fin1. See Supplementary Figure 3g for details of the scheme used for each of the three identical cultures used to generate the samples and Supplmentary Figure 3h for the phenotypic characterisation of one of the three cultures. Samples from asynchronous cultures are run in the left lane in each case to provide a reference standard. (e) Sid2 immunoprecipitates were isolated from asynchronous cultures and employed in in vitro kinase assays utilising 32 PγATP and either recombinant Fin1.KD or casein as indicated. Plots show activity per unit protein (i.e. specific activity) f) Blots with the indicated antibodies of in vitro kinase assays in which the indicated forms of Sid2 were isolated from the respective strains and combined with recombinant Fin1.KDnHis. (g) The incorporation of 32 P into casein from 32 PγATP was used to monitor Sid2 activity in size selected wild type cultures. (h) Sid2 immunoprecipitates were processed as for panel g with the exception that the shorter Fin1.FP1 (non-catalytic C terminal domain 21 ) was used as a substrate and the F1S698P antibody was used to develop the assay with the secondary reagent BCIP. The loading of the Sid2.250 36°C sample in the second to last lane was four times that in other lanes to ensure that the basal level dictated by the reduced level of Sid2.250 protein in the 36°C sample was representative of the reference point for normalisation.
Figure Legend Snippet: Sid2 phosphorylation of Fin1 on serines 377, 526 and 698 promotes Fin1 activity in G2 phase before a peak of each kinase activity accompanies mitotic progression (a, b) Fin1 Kinase assays from size selected cultures in which His tagged Fin1.KD was labeled with 32 PγATP to quantitate activity that is plotted alongside the septation profile. (a) wild type (b) Small G2 sid2.as4 skp1.A4 cells were isolated from a culture grown at 25°C and immediately shifted to 36°C to inactivate Skp1 (and so preserve activated Fin1) at t=0. The culture was split in two and 20 μM 3-MB-PP1 (left assay) or solvent alone (right assay) added after the first division at t = 180. (c, d) Fin1 immunoprecipitates from asynchronous skp1.A4 (c) or cell size selected sid2.as4 skp1.A4 (d) cultures were split in two and probed with antibodies that recognise the indicated phosphorylation sites or polyclonal antibodies that recognise the non-catalytic domain of Fin1. See Supplementary Figure 3g for details of the scheme used for each of the three identical cultures used to generate the samples and Supplmentary Figure 3h for the phenotypic characterisation of one of the three cultures. Samples from asynchronous cultures are run in the left lane in each case to provide a reference standard. (e) Sid2 immunoprecipitates were isolated from asynchronous cultures and employed in in vitro kinase assays utilising 32 PγATP and either recombinant Fin1.KD or casein as indicated. Plots show activity per unit protein (i.e. specific activity) f) Blots with the indicated antibodies of in vitro kinase assays in which the indicated forms of Sid2 were isolated from the respective strains and combined with recombinant Fin1.KDnHis. (g) The incorporation of 32 P into casein from 32 PγATP was used to monitor Sid2 activity in size selected wild type cultures. (h) Sid2 immunoprecipitates were processed as for panel g with the exception that the shorter Fin1.FP1 (non-catalytic C terminal domain 21 ) was used as a substrate and the F1S698P antibody was used to develop the assay with the secondary reagent BCIP. The loading of the Sid2.250 36°C sample in the second to last lane was four times that in other lanes to ensure that the basal level dictated by the reduced level of Sid2.250 protein in the 36°C sample was representative of the reference point for normalisation.

Techniques Used: Activity Assay, Labeling, Isolation, In Vitro, Recombinant

Fin1 kinase is destroyed twice each cell cycle in a Cullin, Fin1 and Sid2 dependent manner (a, b, d, e, h-j) Fin1 levels were normalised to those of Cdc2 kinase in the same lane on the same blot and plotted against time as cells transit the cell cycle (for images of blots see Supplementary Figure 1b ). (a) Fin1 levels declined at two points in wild type cultures; mid-G2 (grey arrow “G2”) and during septation (open arrow “C”). Destruction was seen irrespective of whether the culture was maintained at 25°C throughout the experiment, or shifted to 36°C immediately after size selection ( Supplementary Figure 4c ). (b) Oscillations in Fin1 levels were not seen after synchronised skp1.A4 cultures were shifted to 36°C immediately after size selection at 25°C to inactivate Skp1. (c, g) Normalised Fin1 levels in blots of asynchronous or cdc25.22 arrested double mutant cultures reveal three fold increases in Fin1 levels in the fin1.K33RN165A “kinase dead” and sid2.250 backgrounds. (d) Fin1 levels did not fluctuate as fin1.K33RN165A cultures transited a synchronised cell cycle. (e) Strikingly the levels of both the inactive fin1.K33RN165A protein and the GFP tagged wild type protein oscillate as cells transit the cell cycle when a wild type Fin1.GFP fusion protein was constitutively expressed within the same cells. (f) Fin1 immunoprecipitates from asynchronous cells were employed in kinase assays that used recombinant or casein as substrates. (g) Left: 210 and 240 mins refers to the duration of incubation at 36°C to inactivate and arrest cell cycle progression at the G2/M boundary. Right: FACS profiles of DNA content demonstrate G2 arrest in all strains. (h-j) Assessing the impact of Sid2/Mob1 function upon Fin1 levels in size selected synchronised cultures. (h, j) sid2.250 and mob1.E9 cultures were maintained at 25°C during transit through the first cell division before a portion of the culture was shifted to 36°C to inactivate the kinase/regulatory subunit. (i) A sid2.as4 culture was split into three after the first wave of septation was complete ( Supplementary Figure 1c ) and either nothing, methanol or 3-MB-PP1 in methanol were added to a final concentration of 20μM at time point 190.
Figure Legend Snippet: Fin1 kinase is destroyed twice each cell cycle in a Cullin, Fin1 and Sid2 dependent manner (a, b, d, e, h-j) Fin1 levels were normalised to those of Cdc2 kinase in the same lane on the same blot and plotted against time as cells transit the cell cycle (for images of blots see Supplementary Figure 1b ). (a) Fin1 levels declined at two points in wild type cultures; mid-G2 (grey arrow “G2”) and during septation (open arrow “C”). Destruction was seen irrespective of whether the culture was maintained at 25°C throughout the experiment, or shifted to 36°C immediately after size selection ( Supplementary Figure 4c ). (b) Oscillations in Fin1 levels were not seen after synchronised skp1.A4 cultures were shifted to 36°C immediately after size selection at 25°C to inactivate Skp1. (c, g) Normalised Fin1 levels in blots of asynchronous or cdc25.22 arrested double mutant cultures reveal three fold increases in Fin1 levels in the fin1.K33RN165A “kinase dead” and sid2.250 backgrounds. (d) Fin1 levels did not fluctuate as fin1.K33RN165A cultures transited a synchronised cell cycle. (e) Strikingly the levels of both the inactive fin1.K33RN165A protein and the GFP tagged wild type protein oscillate as cells transit the cell cycle when a wild type Fin1.GFP fusion protein was constitutively expressed within the same cells. (f) Fin1 immunoprecipitates from asynchronous cells were employed in kinase assays that used recombinant or casein as substrates. (g) Left: 210 and 240 mins refers to the duration of incubation at 36°C to inactivate and arrest cell cycle progression at the G2/M boundary. Right: FACS profiles of DNA content demonstrate G2 arrest in all strains. (h-j) Assessing the impact of Sid2/Mob1 function upon Fin1 levels in size selected synchronised cultures. (h, j) sid2.250 and mob1.E9 cultures were maintained at 25°C during transit through the first cell division before a portion of the culture was shifted to 36°C to inactivate the kinase/regulatory subunit. (i) A sid2.as4 culture was split into three after the first wave of septation was complete ( Supplementary Figure 1c ) and either nothing, methanol or 3-MB-PP1 in methanol were added to a final concentration of 20μM at time point 190.

Techniques Used: Selection, Mutagenesis, Recombinant, Incubation, FACS, Concentration Assay

Inhibition of Sid2 or Fin1 delays mitotic commitment (a) 3-MB-PP1 was added to asynchronous cultures of wild type, fin1.as3 and sid2.as4 cells and the mitotic index monitored by anti-α-tubulin immunofluorescence at the indicated times. The analogue transiently inhibited mitotic commitment of fin1.as3 and sid2.as4 but not wild type cells. (b-d) Wild type, fin1.as3 and sid2.as4 cultures were synchronised with respect to cell cycle progression by size selection and split into three equal cultures after the first round of septation. Methanol (MeOH), or 3-MB-PP1 (to a final concentration of 20 μM) in MeOH were added to two of these sub-cultures at 160 minutes. Commitment to mitosis was monitored by the spindle index or phospho-histone H3 reactivity, as indicated. Addition of solvent alone had no impact upon cell cycle progression while addition of analogue in solvent delayed mitotic commitment in fin1.as3 and sid2.as4 but had no impact upon wild type cells.
Figure Legend Snippet: Inhibition of Sid2 or Fin1 delays mitotic commitment (a) 3-MB-PP1 was added to asynchronous cultures of wild type, fin1.as3 and sid2.as4 cells and the mitotic index monitored by anti-α-tubulin immunofluorescence at the indicated times. The analogue transiently inhibited mitotic commitment of fin1.as3 and sid2.as4 but not wild type cells. (b-d) Wild type, fin1.as3 and sid2.as4 cultures were synchronised with respect to cell cycle progression by size selection and split into three equal cultures after the first round of septation. Methanol (MeOH), or 3-MB-PP1 (to a final concentration of 20 μM) in MeOH were added to two of these sub-cultures at 160 minutes. Commitment to mitosis was monitored by the spindle index or phospho-histone H3 reactivity, as indicated. Addition of solvent alone had no impact upon cell cycle progression while addition of analogue in solvent delayed mitotic commitment in fin1.as3 and sid2.as4 but had no impact upon wild type cells.

Techniques Used: Inhibition, Immunofluorescence, Selection, Concentration Assay

Sid2 and Fin1 target Cdr1/Cdr2/Pom1 to control mitotic commitment via Wee1 (a) Cartoon detailing the analogue washout approach. (b-f) 5 hours after the addition of 3-MB-PP1 to early log phase cultures of the indicated strains cells were filtered from the culture and re-suspended at the same density in growth medium that contained no inhibitor. Restoration of Sid2 and Fin1 function induced a burst of mitosis. Importantly, restoration of Sid2 function failed to induce mitotic commitment when Fin1 kinase was inactivated by the fin1.KD mutations (b). (c) Restoring Sid2 activity in strains harbouring mutation of the candidate Sid2 phosphorylation sites in Fin1 suggests that Sid2 can activate Fin1 by phosphorylating the serine at either 377, 526 or 698. Both Sid2 and Fin1 were able to induce mitosis when the cdc2.3w mutation compromised sensitivity to Cdc25, but not when the cdc2.1w mutation compromised Wee1 inhibition of Cdc2 (d), or the functions of Pom1, Cdr1 or Cdr2 are ablated (e, f). (g) Pom1.GFP signals in the indicated strain backgrounds. h) A cartoon depicting the model for G2/M control by Sid2/Fin1. P represents phosphorylation, while Ub represents Ubiquitin conjugation. Fin1 is activated in G2 by phosphorylation by Sid2. This promotes mitotic commitment via modulation of the Geometry network, however, the exact mechanism remains to be determined. Activated Fin1 promotes its own destruction, thereby limiting its activity temporally. Our current lack of understanding of the means by which Fin1 regulates the Pom1/Cdr1/Cdr2/Wee1 cell Geometry Network is represented by incorporating all members of this pathway that are required for Fin1 to regulate mitotic commitment within a single box. The question mark to the left of this box reflects our lack of knowledge as to whether it is the Sid2 or the auto-phosphorylated form of Fin1 that is responsible for the control of the Cell Geometry Network. The question mark beneath Sid1/Cdc14 ref lects our ignorance as to the nature of the cue in G2 phase that triggers this pathway. See text for further details.
Figure Legend Snippet: Sid2 and Fin1 target Cdr1/Cdr2/Pom1 to control mitotic commitment via Wee1 (a) Cartoon detailing the analogue washout approach. (b-f) 5 hours after the addition of 3-MB-PP1 to early log phase cultures of the indicated strains cells were filtered from the culture and re-suspended at the same density in growth medium that contained no inhibitor. Restoration of Sid2 and Fin1 function induced a burst of mitosis. Importantly, restoration of Sid2 function failed to induce mitotic commitment when Fin1 kinase was inactivated by the fin1.KD mutations (b). (c) Restoring Sid2 activity in strains harbouring mutation of the candidate Sid2 phosphorylation sites in Fin1 suggests that Sid2 can activate Fin1 by phosphorylating the serine at either 377, 526 or 698. Both Sid2 and Fin1 were able to induce mitosis when the cdc2.3w mutation compromised sensitivity to Cdc25, but not when the cdc2.1w mutation compromised Wee1 inhibition of Cdc2 (d), or the functions of Pom1, Cdr1 or Cdr2 are ablated (e, f). (g) Pom1.GFP signals in the indicated strain backgrounds. h) A cartoon depicting the model for G2/M control by Sid2/Fin1. P represents phosphorylation, while Ub represents Ubiquitin conjugation. Fin1 is activated in G2 by phosphorylation by Sid2. This promotes mitotic commitment via modulation of the Geometry network, however, the exact mechanism remains to be determined. Activated Fin1 promotes its own destruction, thereby limiting its activity temporally. Our current lack of understanding of the means by which Fin1 regulates the Pom1/Cdr1/Cdr2/Wee1 cell Geometry Network is represented by incorporating all members of this pathway that are required for Fin1 to regulate mitotic commitment within a single box. The question mark to the left of this box reflects our lack of knowledge as to whether it is the Sid2 or the auto-phosphorylated form of Fin1 that is responsible for the control of the Cell Geometry Network. The question mark beneath Sid1/Cdc14 ref lects our ignorance as to the nature of the cue in G2 phase that triggers this pathway. See text for further details.

Techniques Used: Activity Assay, Mutagenesis, Inhibition, Conjugation Assay

29) Product Images from "Cardioprotective effects of iron chelator HAPI and ROS-activated boronate prochelator BHAPI against catecholamine-induced oxidative cellular injury"

Article Title: Cardioprotective effects of iron chelator HAPI and ROS-activated boronate prochelator BHAPI against catecholamine-induced oxidative cellular injury

Journal: Toxicology

doi: 10.1016/j.tox.2016.10.004

Intracellular Fe-chelating efficiency of Fe chelator HAPI and prochelator BHAPI Effects of 24h-preoxidized CA isoprenaline (oxISO) or epinephrine (oxEPI) on prochelator BHAPI inside H9c2 cells were determined during 10 min with Calcein-AM assay. (A, C) Time course of BHAPI activation to effective chelator with (A) oxISO or (C) oxEPI. (B, D) Efficiency of prochelator activation to effective chelator with (B) oxISO or (D) oxEPI at time 10 min. Data are presented as means ± S.D. ; n = 4; Statistical significance (ANOVA, p ≤ 0.05): * vs. control, # vs. corresponding Fe chelator.
Figure Legend Snippet: Intracellular Fe-chelating efficiency of Fe chelator HAPI and prochelator BHAPI Effects of 24h-preoxidized CA isoprenaline (oxISO) or epinephrine (oxEPI) on prochelator BHAPI inside H9c2 cells were determined during 10 min with Calcein-AM assay. (A, C) Time course of BHAPI activation to effective chelator with (A) oxISO or (C) oxEPI. (B, D) Efficiency of prochelator activation to effective chelator with (B) oxISO or (D) oxEPI at time 10 min. Data are presented as means ± S.D. ; n = 4; Statistical significance (ANOVA, p ≤ 0.05): * vs. control, # vs. corresponding Fe chelator.

Techniques Used: Calcein AM Assay, Activation Assay

Comparison of cytotoxic effects of Fe chelator HAPI and prochelator BHAPI towards H9c2 cardiomyoblast cell line Cellular viabilities were determined by neutral red uptake assay and expressed as a percentage of the untreated control group. H9c2 cells were incubated with increasing concentrations of tested compounds for 24h (A, B) or 72h (C, D) Data are presented as means ± S.D. ; n = 4; Statistical significance (ANOVA, p ≤ 0.05): * vs. control group.
Figure Legend Snippet: Comparison of cytotoxic effects of Fe chelator HAPI and prochelator BHAPI towards H9c2 cardiomyoblast cell line Cellular viabilities were determined by neutral red uptake assay and expressed as a percentage of the untreated control group. H9c2 cells were incubated with increasing concentrations of tested compounds for 24h (A, B) or 72h (C, D) Data are presented as means ± S.D. ; n = 4; Statistical significance (ANOVA, p ≤ 0.05): * vs. control group.

Techniques Used: Incubation

Cellular morphology and nuclear epifluorescence staining with Hoechst 33342 and PI H9c2 cardiomyoblasts were incubated for 24 h with (A) control medium, (B) 24h-preoxidized EPI (oxEPI) alone, or in combination with studied compound (C) HAPI or (D) BHAPI, when the compound was added to oxEPI immediately before cellular experiment. Scale bars represent 100 μm.
Figure Legend Snippet: Cellular morphology and nuclear epifluorescence staining with Hoechst 33342 and PI H9c2 cardiomyoblasts were incubated for 24 h with (A) control medium, (B) 24h-preoxidized EPI (oxEPI) alone, or in combination with studied compound (C) HAPI or (D) BHAPI, when the compound was added to oxEPI immediately before cellular experiment. Scale bars represent 100 μm.

Techniques Used: Staining, Incubation

Protective effects of HAPI and BHAPI against oxEPI-induced toxicities towards isolated rat neonatal ventricularcardiomyocytes (NVCM) NVCM were incubated for 24 h with studied compounds or with their combination with 24h-preoxidized EPI (oxEPI; 700 μM). (A) Using epifluorescence microscopy, mitochondrial depolarization was assessed after loading with the JC-1 probe (red emission reflects mitochondrial inner membrane potential-dependent accumulation of probe dimers in actively respiring mitochondria, green fluorescence indicates monomers of the probe released into the cytoplasm after mitochondrial depolarization, lack of fluorescence reflects probe release from necrotic or late-stage apoptotic cells). Scale bars represent 50 μm. (B, C) Inherent toxicities of HAPI and BHAPI determined after 24 h incubation with NVCM by measurement of lactate dehydrogenase release. (D, E) Protective effects of HAPI and BHAPI against oxEPI-induced damage on NVCM assessed by measurement of lactate dehydrogenase release after 24 h incubation. Data are presented as means ± S.D.; n = 4; Statistical significance (ANOVA, p ≤ 0.05): * vs. control group, # vs. oxEPI group.
Figure Legend Snippet: Protective effects of HAPI and BHAPI against oxEPI-induced toxicities towards isolated rat neonatal ventricularcardiomyocytes (NVCM) NVCM were incubated for 24 h with studied compounds or with their combination with 24h-preoxidized EPI (oxEPI; 700 μM). (A) Using epifluorescence microscopy, mitochondrial depolarization was assessed after loading with the JC-1 probe (red emission reflects mitochondrial inner membrane potential-dependent accumulation of probe dimers in actively respiring mitochondria, green fluorescence indicates monomers of the probe released into the cytoplasm after mitochondrial depolarization, lack of fluorescence reflects probe release from necrotic or late-stage apoptotic cells). Scale bars represent 50 μm. (B, C) Inherent toxicities of HAPI and BHAPI determined after 24 h incubation with NVCM by measurement of lactate dehydrogenase release. (D, E) Protective effects of HAPI and BHAPI against oxEPI-induced damage on NVCM assessed by measurement of lactate dehydrogenase release after 24 h incubation. Data are presented as means ± S.D.; n = 4; Statistical significance (ANOVA, p ≤ 0.05): * vs. control group, # vs. oxEPI group.

Techniques Used: Isolation, Incubation, Epifluorescence Microscopy, Fluorescence

Process of BHAPI activation to HAPI HPLC analyses of spontaneous degradation of BHAPI and HAPI and of effects of freshly-prepared or 24h-preoxidized EPI. BHAPI or HAPI were incubated for 24 h: (A, D, G) alone, (B, E, H) with EPI or (C, F, I) with oxEPI in (A, B, C) buffered solution (pH 7.4), (D, E, F) serum-free cell-culture medium or (G, H, I) serum-free cell-culture medium with H9c2 cells. Data are presented as means ± S.D. ; n = 4; Statistical significance (ANOVA, p ≤ 0.05): * vs. t 0 .
Figure Legend Snippet: Process of BHAPI activation to HAPI HPLC analyses of spontaneous degradation of BHAPI and HAPI and of effects of freshly-prepared or 24h-preoxidized EPI. BHAPI or HAPI were incubated for 24 h: (A, D, G) alone, (B, E, H) with EPI or (C, F, I) with oxEPI in (A, B, C) buffered solution (pH 7.4), (D, E, F) serum-free cell-culture medium or (G, H, I) serum-free cell-culture medium with H9c2 cells. Data are presented as means ± S.D. ; n = 4; Statistical significance (ANOVA, p ≤ 0.05): * vs. t 0 .

Techniques Used: Activation Assay, High Performance Liquid Chromatography, Incubation, Cell Culture

Comparison of protective effect of HAPI and BHAPI against ISO- and EPI-induced toxicities towards H9c2 cardiomyoblast cell line Cellular viabilities were determined by neutral red uptake assay and expressed as a percentage of the untreated control group. (A) HAPI or (B) BHAPI were added to freshly-prepared CA in serum-free cell-culture medium before the start of 24h cellular experiments; (C) HAPI or (D) BHAPI were added immediately before cellular experiments to CA preoxidized for 24 h in serum-free cell-culture medium; (E) HAPI or (F) BHAPI were preincubated for 24 h together with CA in serum-free cell-culture medium and then added to cells. Data are presented as means ± S.D. ; n = 4; Statistical significance (ANOVA, p ≤ 0.05): * vs. control group, # vs. corresponding CA group.
Figure Legend Snippet: Comparison of protective effect of HAPI and BHAPI against ISO- and EPI-induced toxicities towards H9c2 cardiomyoblast cell line Cellular viabilities were determined by neutral red uptake assay and expressed as a percentage of the untreated control group. (A) HAPI or (B) BHAPI were added to freshly-prepared CA in serum-free cell-culture medium before the start of 24h cellular experiments; (C) HAPI or (D) BHAPI were added immediately before cellular experiments to CA preoxidized for 24 h in serum-free cell-culture medium; (E) HAPI or (F) BHAPI were preincubated for 24 h together with CA in serum-free cell-culture medium and then added to cells. Data are presented as means ± S.D. ; n = 4; Statistical significance (ANOVA, p ≤ 0.05): * vs. control group, # vs. corresponding CA group.

Techniques Used: Cell Culture

Comparison of effects of HAPI and BHAPI on cellular oxidative stress induced by 24h-preoxidized CA (oxCA – EPI and ISO) Intracellular ROS formation was determined by H 2 DCF-DA assay following the 30min treatment of H9c2 cardiomyoblasts with combination of oxCA and studied compounds. Induced intracellular fluorescence was expressed as a percentage of the group treated with oxCA alone. Cells were incubated with various concentrations of (A) HAPI or (B) BHAPI added immediately before cellular experiments to medium with oxCA (60 μM). Data are presented as means ± S.D. ; n = 4; Statistical significance (ANOVA, p ≤ 0.05): * vs. control group, # vs. corresponding CA group.
Figure Legend Snippet: Comparison of effects of HAPI and BHAPI on cellular oxidative stress induced by 24h-preoxidized CA (oxCA – EPI and ISO) Intracellular ROS formation was determined by H 2 DCF-DA assay following the 30min treatment of H9c2 cardiomyoblasts with combination of oxCA and studied compounds. Induced intracellular fluorescence was expressed as a percentage of the group treated with oxCA alone. Cells were incubated with various concentrations of (A) HAPI or (B) BHAPI added immediately before cellular experiments to medium with oxCA (60 μM). Data are presented as means ± S.D. ; n = 4; Statistical significance (ANOVA, p ≤ 0.05): * vs. control group, # vs. corresponding CA group.

Techniques Used: Fluorescence, Incubation

30) Product Images from "Analysis and Confirmation of 1,3-DMAA and 1,4-DMAA in Geranium Plants Using High Performance Liquid Chromatography with Tandem Mass Spectrometry at ng/g Concentrations"

Article Title: Analysis and Confirmation of 1,3-DMAA and 1,4-DMAA in Geranium Plants Using High Performance Liquid Chromatography with Tandem Mass Spectrometry at ng/g Concentrations

Journal: Analytical Chemistry Insights

doi: 10.4137/ACI.S10445

Typical MRM Chromatogram at 20 μg/L each for 1,3 and 1,4-DMAA analytes. Note: The retention times for the 1,3-DMAA diastereomers are 7.53 and 7.83 minutes, and 1,4-DMAA retention time is 8.17 minutes.
Figure Legend Snippet: Typical MRM Chromatogram at 20 μg/L each for 1,3 and 1,4-DMAA analytes. Note: The retention times for the 1,3-DMAA diastereomers are 7.53 and 7.83 minutes, and 1,4-DMAA retention time is 8.17 minutes.

Techniques Used:

A MRM chromatogram of Changzhou S11-1 sample. Notes: The first two peaks are 1,3-DMAA diastereomer pairs with retention times of 7.51 minutes and 7.81 minutes. The 1,4-DMAA peak retention time is 8.15 minutes. The chromatogram is produced using two mass transitions 116/99.7 m/z and 116/57 m/z.
Figure Legend Snippet: A MRM chromatogram of Changzhou S11-1 sample. Notes: The first two peaks are 1,3-DMAA diastereomer pairs with retention times of 7.51 minutes and 7.81 minutes. The 1,4-DMAA peak retention time is 8.15 minutes. The chromatogram is produced using two mass transitions 116/99.7 m/z and 116/57 m/z.

Techniques Used: Produced

A MRM chromatogram of the optimized extraction protocol for Changzhou S11–2 showing the presence of 1,3-DMAA diastereomers (peaks 1 and 2) and 1,4-DMAA (peak 3). Note: The mass transitions used are 116/99.7 m/z and 116/57 m/z.
Figure Legend Snippet: A MRM chromatogram of the optimized extraction protocol for Changzhou S11–2 showing the presence of 1,3-DMAA diastereomers (peaks 1 and 2) and 1,4-DMAA (peak 3). Note: The mass transitions used are 116/99.7 m/z and 116/57 m/z.

Techniques Used:

A typical MRM chromatogram of the Guiyang 2 sample demonstrating the absence of 1,3-DMAA and 1,4-DMAA in the geranium plant. Note: The mass transitions used are 116/99.7 m/z and 116/57 m/z.
Figure Legend Snippet: A typical MRM chromatogram of the Guiyang 2 sample demonstrating the absence of 1,3-DMAA and 1,4-DMAA in the geranium plant. Note: The mass transitions used are 116/99.7 m/z and 116/57 m/z.

Techniques Used:

A MRM chromatogram of Changzhou 3 sample showing the presence of 1,3-DMAA at a lower concentration than 1,4-DMAA. Note: Mass transitions are 116/99.7 m/z and 116/57 m/z.
Figure Legend Snippet: A MRM chromatogram of Changzhou 3 sample showing the presence of 1,3-DMAA at a lower concentration than 1,4-DMAA. Note: Mass transitions are 116/99.7 m/z and 116/57 m/z.

Techniques Used: Concentration Assay

A MRM chromatogram of the Changzhou 1 sample. Notes: The first two peaks are 1,3-DMAA diastereomer pairs with retention times of 7.51 minutes and 7.81 minutes. The 1,4-DMAA peak retention time is 8.15 minutes. The mass transitions used are 116/99.7 m/z and 116/57 m/z.
Figure Legend Snippet: A MRM chromatogram of the Changzhou 1 sample. Notes: The first two peaks are 1,3-DMAA diastereomer pairs with retention times of 7.51 minutes and 7.81 minutes. The 1,4-DMAA peak retention time is 8.15 minutes. The mass transitions used are 116/99.7 m/z and 116/57 m/z.

Techniques Used:

Chemical structures of the stereoisomers of 1,3-DMAA, 1,4-DMAA, and 2-aminoheptane with stereogenic carbons labeled (*) and their respective (R,S) configurations.
Figure Legend Snippet: Chemical structures of the stereoisomers of 1,3-DMAA, 1,4-DMAA, and 2-aminoheptane with stereogenic carbons labeled (*) and their respective (R,S) configurations.

Techniques Used: Labeling

31) Product Images from "Icariin Metabolism by Human Intestinal Microflora"

Article Title: Icariin Metabolism by Human Intestinal Microflora

Journal: Molecules

doi: 10.3390/molecules21091158

MS spectra of icariin and its metabolites. Thermo Fisher Scientific LCQ fleet instrument (Thermo Scientific, Waltham, MA, USA) was used for electrospray ionization mass spectrometry (ESI-MS) analysis. ESI condition: spray voltage, 5.4 kV; sheath gas, 15 arbitrary units; auxiliary gas, five arbitrary units; heated capillary temperature, 275 °C; capillary voltage, 27 V; and tube lens, 100 V.
Figure Legend Snippet: MS spectra of icariin and its metabolites. Thermo Fisher Scientific LCQ fleet instrument (Thermo Scientific, Waltham, MA, USA) was used for electrospray ionization mass spectrometry (ESI-MS) analysis. ESI condition: spray voltage, 5.4 kV; sheath gas, 15 arbitrary units; auxiliary gas, five arbitrary units; heated capillary temperature, 275 °C; capillary voltage, 27 V; and tube lens, 100 V.

Techniques Used: Mass Spectrometry

Proposed metabolic pathway of icariin in human intestine.
Figure Legend Snippet: Proposed metabolic pathway of icariin in human intestine.

Techniques Used:

Time-dependent biotransformation of icariin by ( a ) Streptococcus sp. MRG-ICA-B and ( b ) Blautia sp. MRG-PMF-1.
Figure Legend Snippet: Time-dependent biotransformation of icariin by ( a ) Streptococcus sp. MRG-ICA-B and ( b ) Blautia sp. MRG-PMF-1.

Techniques Used: Peptide Mass Fingerprinting

HPLC chromatogram changes at 270 nm absorption over icariin metabolism by Blautia sp. MRG-PMF1.
Figure Legend Snippet: HPLC chromatogram changes at 270 nm absorption over icariin metabolism by Blautia sp. MRG-PMF1.

Techniques Used: High Performance Liquid Chromatography

HPLC chromatograms of icariin biotransformation products. Each chromatogram was obtained from the different human intestinal microflora. Microflora a , b and c showed icariside II, icaritin and desmethylicaritin formation, respectively, after 48 h.
Figure Legend Snippet: HPLC chromatograms of icariin biotransformation products. Each chromatogram was obtained from the different human intestinal microflora. Microflora a , b and c showed icariside II, icaritin and desmethylicaritin formation, respectively, after 48 h.

Techniques Used: High Performance Liquid Chromatography

32) Product Images from "High-resolution mass spectrometry analysis of protein oxidations and resultant loss of function"

Article Title: High-resolution mass spectrometry analysis of protein oxidations and resultant loss of function

Journal: Biochemical Society transactions

doi: 10.1042/BST0361037

LTQ tandem mass spectrum of the triply oxidized αB-crystallin peptide 57 APSWFDTGLSEMR 69 . The b 4 ion ( m/z 474.2) is 218.1 bigger than the b 3 ion ( m/z 256.1), showing that the Trp residue contains two oxygen atoms (186.1 + 2 × 16). Similarly,
Figure Legend Snippet: LTQ tandem mass spectrum of the triply oxidized αB-crystallin peptide 57 APSWFDTGLSEMR 69 . The b 4 ion ( m/z 474.2) is 218.1 bigger than the b 3 ion ( m/z 256.1), showing that the Trp residue contains two oxygen atoms (186.1 + 2 × 16). Similarly,

Techniques Used:

33) Product Images from "Multiwalled Carbon Nanotube for One-Step Cleanup of 21 Mycotoxins in Corn and Wheat Prior to Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry Analysis"

Article Title: Multiwalled Carbon Nanotube for One-Step Cleanup of 21 Mycotoxins in Corn and Wheat Prior to Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry Analysis

Journal: Toxins

doi: 10.3390/toxins10100409

Effects of three MWCNT sorbents on recovery (%) of mycotoxins in corn. MWCNT, multiwalled carbon nanotube; MWCNT–COOH, carboxylic MWCNT; MWCNT–OH, hydroxyl MWCNT; DON, deoxynivalenol; NIV, nivalenol; AFB 1 , AFB 2 , AFG 1 , AFG 2 , aflatoxins; 15-AcDON, 15-acetyldeoxynivalenol; 3-AcDON, 3-acetyldeoxynivalenol; FUS-X, fusarenon X; DAS, diacetoxyscirpenol; OTA, ochratoxin A; OTB, ochratoxin B; T-2, T-2 toxin; HT-2, HT-2 toxin; NEO, neosolaniol; ZEN, zearalenone; α-ZOL, α-zearalenol; β-ZOL, β-zearalenol; ZAN, zearalanone; α-ZAL, α-zearalanol; β-ZAL, β-zearalanol. Vertical bar represents ± standard error ( n = 3).
Figure Legend Snippet: Effects of three MWCNT sorbents on recovery (%) of mycotoxins in corn. MWCNT, multiwalled carbon nanotube; MWCNT–COOH, carboxylic MWCNT; MWCNT–OH, hydroxyl MWCNT; DON, deoxynivalenol; NIV, nivalenol; AFB 1 , AFB 2 , AFG 1 , AFG 2 , aflatoxins; 15-AcDON, 15-acetyldeoxynivalenol; 3-AcDON, 3-acetyldeoxynivalenol; FUS-X, fusarenon X; DAS, diacetoxyscirpenol; OTA, ochratoxin A; OTB, ochratoxin B; T-2, T-2 toxin; HT-2, HT-2 toxin; NEO, neosolaniol; ZEN, zearalenone; α-ZOL, α-zearalenol; β-ZOL, β-zearalenol; ZAN, zearalanone; α-ZAL, α-zearalanol; β-ZAL, β-zearalanol. Vertical bar represents ± standard error ( n = 3).

Techniques Used: Ziehl-Neelsen Stain

34) Product Images from "Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum"

Article Title: Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum

Journal: Nature Communications

doi: 10.1038/ncomms10111

In vitro binding and functional validation of artemisinin targets. ( a ) Artemisinin specifically interacts with OAT, PyrK, LDH, SpdSyn, SAMS and TCTP as the unlabelled artesunate (25 ×) can compete with the AP1 binding. Heat denaturation reduces the AP1 -labelling level of OAT, suggesting that the interaction of artemisinin with OAT is activity based. ( b ) Dose-dependent labelling of OAT with AP1 (4 h treatment). ( c ) Time-dependent labelling of OAT with AP1 . ( d ) The interaction of artemisinin with OAT may involve thiol and amine groups as IAA (blocking thiol, 30 mM) and NEM (blocking amine, 10 mM) pretreatment (20 min) can reduce binding. ( e , f ) Activated artesunate inhibits the activities of PyrK ( e ) and LDH ( f ) in vitro . Δ, heat denaturation; IAA, iodoacetamide; NEM, N -ethylmaleimide; Conc., concentration. Error bars represent s.d. in three independent replicates in e and f . Full-gel images for panels a – d are shown in Supplementary Fig. 13 .
Figure Legend Snippet: In vitro binding and functional validation of artemisinin targets. ( a ) Artemisinin specifically interacts with OAT, PyrK, LDH, SpdSyn, SAMS and TCTP as the unlabelled artesunate (25 ×) can compete with the AP1 binding. Heat denaturation reduces the AP1 -labelling level of OAT, suggesting that the interaction of artemisinin with OAT is activity based. ( b ) Dose-dependent labelling of OAT with AP1 (4 h treatment). ( c ) Time-dependent labelling of OAT with AP1 . ( d ) The interaction of artemisinin with OAT may involve thiol and amine groups as IAA (blocking thiol, 30 mM) and NEM (blocking amine, 10 mM) pretreatment (20 min) can reduce binding. ( e , f ) Activated artesunate inhibits the activities of PyrK ( e ) and LDH ( f ) in vitro . Δ, heat denaturation; IAA, iodoacetamide; NEM, N -ethylmaleimide; Conc., concentration. Error bars represent s.d. in three independent replicates in e and f . Full-gel images for panels a – d are shown in Supplementary Fig. 13 .

Techniques Used: In Vitro, Binding Assay, Functional Assay, Activity Assay, Blocking Assay, Concentration Assay

35) Product Images from "Selective Chemoprecipitation to Enrich Nitropeptides from Complex Proteomes for Mass Spectrometric Analysis"

Article Title: Selective Chemoprecipitation to Enrich Nitropeptides from Complex Proteomes for Mass Spectrometric Analysis

Journal: Nature protocols

doi: 10.1038/nprot.2014.052

Reaction scheme for the preparation of SPAER. Steps: ( a ) Succinylation of aminopropyl-modified controlled pore glass (CPG-C 3 H 7 -NH 2 ) with succinic anhydride (SA); ( b ) Fmoc-hydrazide formation via N-hydroxysuccinimide active ester in situ generated from
Figure Legend Snippet: Reaction scheme for the preparation of SPAER. Steps: ( a ) Succinylation of aminopropyl-modified controlled pore glass (CPG-C 3 H 7 -NH 2 ) with succinic anhydride (SA); ( b ) Fmoc-hydrazide formation via N-hydroxysuccinimide active ester in situ generated from

Techniques Used: Modification, In Situ, Generated

36) Product Images from "Improved HPLC Method Using 2,3-naphthalenedicarboxaldehyde as Fluorescent Labeling Agent for Quantification of Histamine in Human Immunoglobulin Preparations"

Article Title: Improved HPLC Method Using 2,3-naphthalenedicarboxaldehyde as Fluorescent Labeling Agent for Quantification of Histamine in Human Immunoglobulin Preparations

Journal: Osong Public Health and Research Perspectives

doi: 10.1016/j.phrp.2011.07.003

Derivatization reaction schemes of OPA (A) and NDA (B) with histamine.
Figure Legend Snippet: Derivatization reaction schemes of OPA (A) and NDA (B) with histamine.

Techniques Used:

HPLC chromatograms of different fluorescent labeling agents: (A) OPA; and (B) NDA. Six different concentrations of histamine-containing samples with IS were analyzed and the chromatograms overlapped after each fluorescent agents’ derivatization.
Figure Legend Snippet: HPLC chromatograms of different fluorescent labeling agents: (A) OPA; and (B) NDA. Six different concentrations of histamine-containing samples with IS were analyzed and the chromatograms overlapped after each fluorescent agents’ derivatization.

Techniques Used: High Performance Liquid Chromatography, Labeling

37) Product Images from "New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa"

Article Title: New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa

Journal: Marine Drugs

doi: 10.3390/md13106038

Elution profiles of H. crispa polypeptides at various stages of chromatographic purification. ( A ) Gel filtration chromatography of polypeptides contained in 80% acetone powder on column with Akrilex P-4; ( B ) Subsequent cation-exchange chromatography of active fraction polypeptides ( Figure 1 A, peak 3) on column with cellulose CM-32; ( C ) RP-HPLC performed on Nucleosil C 18 column of polypeptides ( Figure 1 B, peak 4) desalted on an Akrilex P-4 column. Fraction with hemolytic and trypsin inhibitory activities are accentuated by solid and dotted lines, respectively. MALDI-TOF/MS spectrums and molecular masses of HCRG1 ( D ) and HCRG2 ( E ) after RP-HPLC are shown in the inset. Chromatography conditions are described in the Experimental Section (Methods).
Figure Legend Snippet: Elution profiles of H. crispa polypeptides at various stages of chromatographic purification. ( A ) Gel filtration chromatography of polypeptides contained in 80% acetone powder on column with Akrilex P-4; ( B ) Subsequent cation-exchange chromatography of active fraction polypeptides ( Figure 1 A, peak 3) on column with cellulose CM-32; ( C ) RP-HPLC performed on Nucleosil C 18 column of polypeptides ( Figure 1 B, peak 4) desalted on an Akrilex P-4 column. Fraction with hemolytic and trypsin inhibitory activities are accentuated by solid and dotted lines, respectively. MALDI-TOF/MS spectrums and molecular masses of HCRG1 ( D ) and HCRG2 ( E ) after RP-HPLC are shown in the inset. Chromatography conditions are described in the Experimental Section (Methods).

Techniques Used: Purification, Filtration, Chromatography, High Performance Liquid Chromatography, Mass Spectrometry

38) Product Images from "Borneol and Α-asarone as adjuvant agents for improving blood–brain barrier permeability of puerarin and tetramethylpyrazine by activating adenosine receptors"

Article Title: Borneol and Α-asarone as adjuvant agents for improving blood–brain barrier permeability of puerarin and tetramethylpyrazine by activating adenosine receptors

Journal: Drug Delivery

doi: 10.1080/10717544.2018.1516005

The cumulative permeability of PUE and TMP ( n = 3). (A) effects of different concentration of borneol and α-asarone on the PUE cumulative permeability; (B) effects of adenosine receptor inhibitors on the PUE cumulative permeability; (C) effects of different concentration of borneol and α-asarone on the TMP cumulative permeability; (D) effects of adenosine receptor inhibitors on the TMP cumulative permeability. Concentration for PUE and TMP were 50 μM. * p
Figure Legend Snippet: The cumulative permeability of PUE and TMP ( n = 3). (A) effects of different concentration of borneol and α-asarone on the PUE cumulative permeability; (B) effects of adenosine receptor inhibitors on the PUE cumulative permeability; (C) effects of different concentration of borneol and α-asarone on the TMP cumulative permeability; (D) effects of adenosine receptor inhibitors on the TMP cumulative permeability. Concentration for PUE and TMP were 50 μM. * p

Techniques Used: Permeability, Concentration Assay

In vivo time–concentration profiles of PUE and TMP ( n = 6). (A) the concentration–time profiles of PUE in brain tissue homogenate; (B) the concentration–time profiles of PUE in plasma; (C) the concentration–time profiles of TMP in brain tissue homogenate; (D) the concentration–time profiles of TMP in plasma; (E) comparison of PUE delivery into BBB (AUC brain) and in the plasma (AUC plasma); (F) comparison of TMP delivery into BBB (AUC brain) and in the plasma (AUC plasma). Doses orally administrate: PUE (20 mg/kg), TMP (10 mg/kg), Borneol (25 mg/kg) and α-asarone (25 mg/kg). * p
Figure Legend Snippet: In vivo time–concentration profiles of PUE and TMP ( n = 6). (A) the concentration–time profiles of PUE in brain tissue homogenate; (B) the concentration–time profiles of PUE in plasma; (C) the concentration–time profiles of TMP in brain tissue homogenate; (D) the concentration–time profiles of TMP in plasma; (E) comparison of PUE delivery into BBB (AUC brain) and in the plasma (AUC plasma); (F) comparison of TMP delivery into BBB (AUC brain) and in the plasma (AUC plasma). Doses orally administrate: PUE (20 mg/kg), TMP (10 mg/kg), Borneol (25 mg/kg) and α-asarone (25 mg/kg). * p

Techniques Used: In Vivo, Concentration Assay

39) Product Images from "A Fast and Validated Reversed-Phase HPLC Method for Simultaneous Determination of Simvastatin, Atorvastatin, Telmisartan and Irbesartan in Bulk Drugs and Tablet Formulations"

Article Title: A Fast and Validated Reversed-Phase HPLC Method for Simultaneous Determination of Simvastatin, Atorvastatin, Telmisartan and Irbesartan in Bulk Drugs and Tablet Formulations

Journal: Scientia Pharmaceutica

doi: 10.3390/scipharm86010001

Chemical structures of ( A ) Atorvastatin (ATV); ( B ) Simvastatin (SMV); ( C ) Telmisartan (TLN) and ( D ) Irbesartan (IRB).
Figure Legend Snippet: Chemical structures of ( A ) Atorvastatin (ATV); ( B ) Simvastatin (SMV); ( C ) Telmisartan (TLN) and ( D ) Irbesartan (IRB).

Techniques Used:

Calibration curve showing excellent linearity of the method: ( A ) Irbesartan; ( B ) Atorvastatin; ( C ) Telmisartan and ( D ) Simvastatin.
Figure Legend Snippet: Calibration curve showing excellent linearity of the method: ( A ) Irbesartan; ( B ) Atorvastatin; ( C ) Telmisartan and ( D ) Simvastatin.

Techniques Used:

Representative chromatograms of individual analytes in tablet dosage forms. ( A ) Irbesartan; ( B ) Atorvastatin; ( C ) Telmisartan; and ( D ) Simvastatin. Conditions : stationary phase, Symmetry C18 column; mobile phase, 10 mM ammonium acetate buffer (pH 4)–acetonitrile (40:60 v / v ); flow rate, 1 mL/min up to 3.5 min then 2 mL/min; detection, UV 220 nm.
Figure Legend Snippet: Representative chromatograms of individual analytes in tablet dosage forms. ( A ) Irbesartan; ( B ) Atorvastatin; ( C ) Telmisartan; and ( D ) Simvastatin. Conditions : stationary phase, Symmetry C18 column; mobile phase, 10 mM ammonium acetate buffer (pH 4)–acetonitrile (40:60 v / v ); flow rate, 1 mL/min up to 3.5 min then 2 mL/min; detection, UV 220 nm.

Techniques Used: Flow Cytometry

Chromatogram showing excellent separation between irbesartan, atorvastatin, telmisartan and simvastatin. Conditions: stationary phase, Symmetry C18 column; mobile phase, 10 mM ammonium acetate buffer (pH 4)–acetonitrile (40:60 v / v ); flow rate, 1 mL/min up to 3.5 min then 2 mL/min; detection, UV 220 nm.
Figure Legend Snippet: Chromatogram showing excellent separation between irbesartan, atorvastatin, telmisartan and simvastatin. Conditions: stationary phase, Symmetry C18 column; mobile phase, 10 mM ammonium acetate buffer (pH 4)–acetonitrile (40:60 v / v ); flow rate, 1 mL/min up to 3.5 min then 2 mL/min; detection, UV 220 nm.

Techniques Used: Flow Cytometry

40) Product Images from "Liquid chromatography electrospray ionization tandem mass spectrometry analysis method for simultaneous detection of trichloroacetic acid, dichloroacetic acid, S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine"

Article Title: Liquid chromatography electrospray ionization tandem mass spectrometry analysis method for simultaneous detection of trichloroacetic acid, dichloroacetic acid, S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine

Journal: Toxicology

doi: 10.1016/j.tox.2009.06.013

MS/MS product ion spectra of [M-H] - ions of chemical standards. (A) dichloroacetic acid, (B) difluoroacetic acid, (C) trichloroacetic acid, and (D) trifluoroacetic acid.
Figure Legend Snippet: MS/MS product ion spectra of [M-H] - ions of chemical standards. (A) dichloroacetic acid, (B) difluoroacetic acid, (C) trichloroacetic acid, and (D) trifluoroacetic acid.

Techniques Used: Mass Spectrometry

Related Articles

Incubation:

Article Title: Tenascin-R Is an Intrinsic Autocrine Factor for Oligodendrocyte Differentiation and Promotes Cell Adhesion by a SulfatideMediated Mechanism
Article Snippet: .. Wells subsequently were washed with cold PBS, and the binding of biotinylated (with N -hydroxysuccinimide biotin ester, Sigma, Deisenhofen, Germany) TN-R 160 and TN-R 180 (2 μg/ml in PBS containing 1% BSA, 100 μl/well) was determined after 2 hr of incubation at 37°C by using HRP-conjugated streptavidin (Sigma; diluted in PBS containing 1% BSA, 100 μl/well) for detection. .. Similar results were obtained when unlabeled TN-R detected by polyclonal antibodies to TN-R and HRP-conjugated goat anti-rabbit IgG antibodies were used for binding studies.

other:

Article Title: Antitumor effect of a Pt-loaded nanocomposite based on graphene quantum dots combats hypoxia-induced chemoresistance of oral squamous cell carcinoma
Article Snippet: Materials and agents Cis-Pt(NH3 )2 Cl2 (purity > 98%), 4arm-PEG-Amine, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), O-phenylenediamine (OPDA), and dimethylformamide (DMF) were purchased from Sigma-Aldrich Co (St Louis, MO, USA).

Article Title: Implantation of VEGF-functionalized cell-free vascular grafts: regenerative and immunological response
Article Snippet: The SIS was then allowed to dry on the mandrel for 2 h at room temperature before it was cross-linked in a solution containing 20 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (MilliporeSigma, Burlington, MA, USA), 10 mM N-hydroxysuccinimide (MilliporeSigma), in 50 mM 2-ethanesulfonic acid buffer (pH 4.5) for 2 h at room temperature with gentle rocking.

Droplet Countercurrent Chromatography:

Article Title: A novel paclitaxel-loaded poly(d,l-lactide-co-glycolide)-Tween 80 copolymer nanoparticle overcoming multidrug resistance for lung cancer treatment
Article Snippet: .. D-alpha tocopheryl polyethyleneglycol 1000 succinate (TPGS), N ,N ′-dicyclohexylcarbodiimide (DCC), Tween 80, N -hydroxysuccinimide (NHS), propidium iodide (PI), RNase A, Coumarin-6 (C-6), and trypsin-EDTA were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). .. RPMI 1640 medium, penicillin–streptomycin, fetal bovine serum, and phosphate-buffered saline (PBS) were purchased from Hyclone (Logan, UT, USA).

BIA-KA:

Article Title: Selective Chemoprecipitation to Enrich Nitropeptides from Complex Proteomes for Mass Spectrometric Analysis
Article Snippet: .. Acetic acid (AcOH; Sigma-Aldrich, cat. no. 338826) Acetonitrile (ACN; EMD, cat. no. AX0145) Aminopropyl-modified controlled pore glass particle, size 120-200 mesh, 0.2 mmol/g loading (CPG-C3 H7 -NH2 ; Sigma-Aldrich, cat. no. 27791) Ammonium bicarbonate (NH4 HCO3; Sigma-Aldrich, cat. no. A6141) Ammonium hydroxide solution (NH4 OH; Sigma-Aldrich, 338818) BCA assay (Thermo Scientific, cat. no. 23225) Custom-synthesized nitropeptide(s) (Synthetic BioMolecules) – optional, for quality control of SPAER and workflow, as well as hit validation D13 CDO, (Cambridge Isotope Laboratories, cat. no. CDLM-4599-1) – optional, “heavy” formaldehyde for differential dimethylation in quantitative nitroproteomics DCDO (Sigma-Aldrich, cat. no. 492620) – optional, “intermediate” formaldehyde for differential dimethylation in quantitative nitroproteomics Dichloromethane (DCM; Sigma-Aldrich, cat. no. 650463) N,N-Diisopropylcarbodiimide (DIC; Sigma-Aldrich, cat. no. D125407) N-Hydroxysuccinimide (HOSu; Sigma-Aldrich, cat. no. 56480) N,N-Dimethylformamide (DMF; Sigma-Aldrich, cat. no. 227056) N,N-Diisopropyl ethylamine (DIPEA; Sigma-Aldrich, cat. no. 387649) Dithiothreitol (DTT; Sigma-Aldrich, cat. no. D9779) 9-Fluorenylmethyl carbazate (Fmoc-NH-NH2; Sigma-Aldrich, cat. no. 46917) Formaldehyde (HCHO; Sigma-Aldrich, cat. no. F8775) Formic acid (FA; Sigma-Aldrich, cat. no. 14265) 4-Formylbenzoic acid N-hydroxysuccinimide ester (4FB-OSu; Sigma-Aldrich, cat. No. 40923). .. Iodoacetamide (IAA; Sigma-Aldrich, cat. no. I1149) Methanol (MeOH; Sigma-Aldrich, cat. no. 34860) Phosphate buffer solution (PBS; Sigma-Aldrich, cat. no. P5244) Piperidine (Sigma-Aldrich, cat. No. 411027) Sequencing-grade modified porcine trypsin (Promega, cat. no. V5111) Sodium acetate (Sigma-Aldrich, cat. no. S8750) Sodium chloride (NaCl; Sigma-Aldrich, cat. no. S7653) Sodium cyanoborodeuteride (NaBD3 CN; Sigma-Aldrich, cat. no.190020) – optional, “heavy” reductant for differential dimethylation in quantitative proteomics Sodium cyanoborohydride (NaBH3 CN; Sigma-Aldrich, cat. no.156159) Sodium dithionite (Na2 S2 O4 ; Sigma-Aldrich, cat. no. 157953) Sodium hydroxide solution (NaOH; Sigma-Aldrich, cat. no. 415413) Succinic anhydride (SA; a.k.a. dihydro-2,5-furandione, Sigma-Aldrich, cat. no. 239690) Tetranitromethane (TNM; Sigma-Aldrich, cat. no. ) – optional, for quality control of SPAER and workflow Trifluoroacetic acid (TFA; Sigma-Aldrich, cat. no. 302031) Urea (Sigma-Aldrich, cat. no. U6504) Universal Proteomics Standard Set (Sigma-Aldrich, cat. no UPS1) – optional, for quality control of SPAER and workflow Water (H2 O, HPLC grade; Fisher Scientific, cat. no. W5-4)

Binding Assay:

Article Title: Tenascin-R Is an Intrinsic Autocrine Factor for Oligodendrocyte Differentiation and Promotes Cell Adhesion by a SulfatideMediated Mechanism
Article Snippet: .. Wells subsequently were washed with cold PBS, and the binding of biotinylated (with N -hydroxysuccinimide biotin ester, Sigma, Deisenhofen, Germany) TN-R 160 and TN-R 180 (2 μg/ml in PBS containing 1% BSA, 100 μl/well) was determined after 2 hr of incubation at 37°C by using HRP-conjugated streptavidin (Sigma; diluted in PBS containing 1% BSA, 100 μl/well) for detection. .. Similar results were obtained when unlabeled TN-R detected by polyclonal antibodies to TN-R and HRP-conjugated goat anti-rabbit IgG antibodies were used for binding studies.

Molecular Weight:

Article Title: Synthesis of composite magnetic nanoparticles Fe3O4 with alendronate for osteoporosis treatment
Article Snippet: .. Ferrous chloride tetrahydrate (FeCl2 ⋅4H2 O), ferrous chloride hexahydrate (FeCl3 ⋅6H2 O), Dex ((C6 H10 O5 )n , molecular weight [MW] ~40,000 Da), hydrazine (N2 H4 ), sodium hydroxide (NaOH), sodium chloroacetate (C2 H2 ClO2 ⋅Na), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride (C8 H17 N3 ⋅HCl), N -hydroxysuccinimide (C4 H5 NO3 ; NHS), and alendronate sodium trihydrant (C4 H12 NO7 P2 Na⋅3H2 O) were all purchased from Sigma-Aldrich Co. (St Louis, MO, USA) and then used, as received, to synthesize the composite nanoparticles. .. Reagents used in cell culture include albumin solution from bovine serum (Sigma-Aldrich Co.), ascorbic acid (C6 H8 O6 ; Sigma-Aldrich Co.), Dulbecco’s Modified Eagle’s Medium (DMEM; high glucose with L-glutamine and pyridoxine hydrochloride; GibcoÒ; Thermo Fisher Scientific, Waltham, MA, USA), alpha-minimum essential medium (GibcoÒ; Thermo Fisher Scientific), sodium bicarbonate (NaHCO3 ; Sigma-Aldrich Co.), hydrochloric acid (HCl; 36.5%–38%; J.T.

Article Title: Construction of magnetic-carbon-quantum-dots-probe-labeled apoferritin nanocages for bioimaging and targeted therapy
Article Snippet: .. Ferritin from equine spleen, FA, citric acid, polyethylenimine (PEI; molecular weight =2,500), diethylene glycol, ethylenediamine, formamide, N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl-N′-ethylcarbodiimide) hydrochloride (EDC-HCl), and dimethyl sulfoxide were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). .. 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle’s Medium cell culture medium, penicillin, streptomycin, fetal bovine serum, and heparin sodium were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

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  • 96
    Millipore imatinib hydrochloride salt
    The comparison of time–activity curves of the (A) heart, (B) lungs, (C) kidneys, (D) spleen, (E) liver and (F) gallbladder before and after pretreatment with 32 mg of <t>imatinib</t> administration by intravenous injection. Time–activity curves
    Imatinib Hydrochloride Salt, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/imatinib hydrochloride salt/product/Millipore
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    imatinib hydrochloride salt - by Bioz Stars, 2020-07
    96/100 stars
      Buy from Supplier

    99
    Millipore jnk inhibitor v
    POH-induced apoptosis in U251 cells. U251 in the control condition (Control, A) . The cells were treated with 0.5 mM POH (POH, B) and 0.5 mM POH plus 0.5 μM <t>JNK</t> <t>inhibitor</t> V (POH + IJNK, C) . After 24 hours, the cells were immunostained for cleaved caspase-3 and the number of positive cells was analyzed (D) . Whereas POH induced cell apoptosis, the addition of the JNK inhibitor completely inhibited this effect. The addition of DMSO or JNK inhibitor V alone had no effect on cell death. Scale bar: 20 μm.*P
    Jnk Inhibitor V, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/jnk inhibitor v/product/Millipore
    Average 99 stars, based on 39 article reviews
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    jnk inhibitor v - by Bioz Stars, 2020-07
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    85
    Millipore as 601245
    POH-induced apoptosis in U251 cells. U251 in the control condition (Control, A) . The cells were treated with 0.5 mM POH (POH, B) and 0.5 mM POH plus 0.5 μM <t>JNK</t> <t>inhibitor</t> V (POH + IJNK, C) . After 24 hours, the cells were immunostained for cleaved caspase-3 and the number of positive cells was analyzed (D) . Whereas POH induced cell apoptosis, the addition of the JNK inhibitor completely inhibited this effect. The addition of DMSO or JNK inhibitor V alone had no effect on cell death. Scale bar: 20 μm.*P
    As 601245, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/as 601245/product/Millipore
    Average 85 stars, based on 1 article reviews
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    Image Search Results


    The comparison of time–activity curves of the (A) heart, (B) lungs, (C) kidneys, (D) spleen, (E) liver and (F) gallbladder before and after pretreatment with 32 mg of imatinib administration by intravenous injection. Time–activity curves

    Journal: Nuclear medicine and biology

    Article Title: Synthesis and positron emission tomography studies of carbon-11-labeled imatinib (Gleevec)

    doi: 10.1016/j.nucmedbio.2006.11.004

    Figure Lengend Snippet: The comparison of time–activity curves of the (A) heart, (B) lungs, (C) kidneys, (D) spleen, (E) liver and (F) gallbladder before and after pretreatment with 32 mg of imatinib administration by intravenous injection. Time–activity curves

    Article Snippet: The excess amount of hydrochloric acid was removed by coevaporation with acetonitrile to give imatinib hydrochloride salt, which was dissolved in 5 ml of sterile water and passed through a Millipore filter.

    Techniques: Activity Assay, Injection

    Structure of imatinib (the active ingredient of Gleevec).

    Journal: Nuclear medicine and biology

    Article Title: Synthesis and positron emission tomography studies of carbon-11-labeled imatinib (Gleevec)

    doi: 10.1016/j.nucmedbio.2006.11.004

    Figure Lengend Snippet: Structure of imatinib (the active ingredient of Gleevec).

    Article Snippet: The excess amount of hydrochloric acid was removed by coevaporation with acetonitrile to give imatinib hydrochloride salt, which was dissolved in 5 ml of sterile water and passed through a Millipore filter.

    Techniques:

    Synthetic scheme of norimatinib, imatinib and [ N - 11 C-methyl]imatinib.

    Journal: Nuclear medicine and biology

    Article Title: Synthesis and positron emission tomography studies of carbon-11-labeled imatinib (Gleevec)

    doi: 10.1016/j.nucmedbio.2006.11.004

    Figure Lengend Snippet: Synthetic scheme of norimatinib, imatinib and [ N - 11 C-methyl]imatinib.

    Article Snippet: The excess amount of hydrochloric acid was removed by coevaporation with acetonitrile to give imatinib hydrochloride salt, which was dissolved in 5 ml of sterile water and passed through a Millipore filter.

    Techniques:

    PET image of baboon brain with [ N - 11 C-methyl]imatinib. Summed frames over 90 min after the injection of 3.9 mCi of [ N - 11 ]. The anesthesia

    Journal: Nuclear medicine and biology

    Article Title: Synthesis and positron emission tomography studies of carbon-11-labeled imatinib (Gleevec)

    doi: 10.1016/j.nucmedbio.2006.11.004

    Figure Lengend Snippet: PET image of baboon brain with [ N - 11 C-methyl]imatinib. Summed frames over 90 min after the injection of 3.9 mCi of [ N - 11 ]. The anesthesia

    Article Snippet: The excess amount of hydrochloric acid was removed by coevaporation with acetonitrile to give imatinib hydrochloride salt, which was dissolved in 5 ml of sterile water and passed through a Millipore filter.

    Techniques: Positron Emission Tomography, Injection

    PET image of the torso of the anesthetized baboon with [ N - 11 C-methyl]imatinib. PET images (summation of frames over 90 min) of the baboon torso after the injection of 4.71 mCi of [ N - 11 C-methyl]imatinib showing the accumulation of carbon-11 in the heart

    Journal: Nuclear medicine and biology

    Article Title: Synthesis and positron emission tomography studies of carbon-11-labeled imatinib (Gleevec)

    doi: 10.1016/j.nucmedbio.2006.11.004

    Figure Lengend Snippet: PET image of the torso of the anesthetized baboon with [ N - 11 C-methyl]imatinib. PET images (summation of frames over 90 min) of the baboon torso after the injection of 4.71 mCi of [ N - 11 C-methyl]imatinib showing the accumulation of carbon-11 in the heart

    Article Snippet: The excess amount of hydrochloric acid was removed by coevaporation with acetonitrile to give imatinib hydrochloride salt, which was dissolved in 5 ml of sterile water and passed through a Millipore filter.

    Techniques: Positron Emission Tomography, Injection

    (A) Time–activity curve at baseline and after imatinib pretreatment for [ N - 11 C-methyl]imatinib in plasma showing the first 5 min and (B) the plasma integral over 90 min. Pretreatment delayed [ N - 11 C-methyl]imatinib peak and reduced its accumulated

    Journal: Nuclear medicine and biology

    Article Title: Synthesis and positron emission tomography studies of carbon-11-labeled imatinib (Gleevec)

    doi: 10.1016/j.nucmedbio.2006.11.004

    Figure Lengend Snippet: (A) Time–activity curve at baseline and after imatinib pretreatment for [ N - 11 C-methyl]imatinib in plasma showing the first 5 min and (B) the plasma integral over 90 min. Pretreatment delayed [ N - 11 C-methyl]imatinib peak and reduced its accumulated

    Article Snippet: The excess amount of hydrochloric acid was removed by coevaporation with acetonitrile to give imatinib hydrochloride salt, which was dissolved in 5 ml of sterile water and passed through a Millipore filter.

    Techniques: Activity Assay

    Time–activity curves for two different anesthetized baboons (A and B) that each received two injections of [ N - 11 C-methyl]imatinib in the brain through the urinary bladder.

    Journal: Nuclear medicine and biology

    Article Title: Synthesis and positron emission tomography studies of carbon-11-labeled imatinib (Gleevec)

    doi: 10.1016/j.nucmedbio.2006.11.004

    Figure Lengend Snippet: Time–activity curves for two different anesthetized baboons (A and B) that each received two injections of [ N - 11 C-methyl]imatinib in the brain through the urinary bladder.

    Article Snippet: The excess amount of hydrochloric acid was removed by coevaporation with acetonitrile to give imatinib hydrochloride salt, which was dissolved in 5 ml of sterile water and passed through a Millipore filter.

    Techniques: Activity Assay

    POH-induced apoptosis in U251 cells. U251 in the control condition (Control, A) . The cells were treated with 0.5 mM POH (POH, B) and 0.5 mM POH plus 0.5 μM JNK inhibitor V (POH + IJNK, C) . After 24 hours, the cells were immunostained for cleaved caspase-3 and the number of positive cells was analyzed (D) . Whereas POH induced cell apoptosis, the addition of the JNK inhibitor completely inhibited this effect. The addition of DMSO or JNK inhibitor V alone had no effect on cell death. Scale bar: 20 μm.*P

    Journal: Molecular Cancer

    Article Title: Na/K-ATPase as a target for anticancer drugs: studies with perillyl alcohol

    doi: 10.1186/s12943-015-0374-5

    Figure Lengend Snippet: POH-induced apoptosis in U251 cells. U251 in the control condition (Control, A) . The cells were treated with 0.5 mM POH (POH, B) and 0.5 mM POH plus 0.5 μM JNK inhibitor V (POH + IJNK, C) . After 24 hours, the cells were immunostained for cleaved caspase-3 and the number of positive cells was analyzed (D) . Whereas POH induced cell apoptosis, the addition of the JNK inhibitor completely inhibited this effect. The addition of DMSO or JNK inhibitor V alone had no effect on cell death. Scale bar: 20 μm.*P

    Article Snippet: Cell death assay U87 and U251 cells were pretreated for 30 minutes with JNK inhibitor V [1,3-Benzothiazol-2-yl-(2-((2-(3-pyridinyl)ethyl)amino)-4-pyrimidinyl)acetonitrile; Calbiochem], an inhibitor of JNK1/2 activation, before treatment with 0.5 mM POH and 0.5 mM POH plus 0.5 μM JNK inhibitor V. After 24 hours of incubation, the cells were suspended in annexin and propidium iodide binding buffer as specified in the TACS Annexin V-FITC apoptosis detection kit (R & D Systems).

    Techniques:

    The effects of JNK inhibition on the induction of cell death by POH in U251 cells. Before treatment, U251 cells were incubated without (A and B) or with (C and D) JNK inhibitor V (0.5 μM) for 30 minutes. The cells were treated with 0.1% DMSO (A) , 0.5 mM POH (B) 0.1% DMSO plus JNK inhibitor V (C) , and 0.5 mM POH plus JNK inhibitor V (D) . After 24 hours of incubation, the cells were stained with annexin V-FITC and propidium iodide and analyzed by flow cytometry. Figure 7E represents the percentage of dead cells as indicated by early apoptosis and late apoptosis or necrosis (right lower quadrant + right upper quadrant, respectively), which was calculated from the data shown in Figures 7A-D. The data were expressed as the means ± SD from at least three different experiments. ***p

    Journal: Molecular Cancer

    Article Title: Na/K-ATPase as a target for anticancer drugs: studies with perillyl alcohol

    doi: 10.1186/s12943-015-0374-5

    Figure Lengend Snippet: The effects of JNK inhibition on the induction of cell death by POH in U251 cells. Before treatment, U251 cells were incubated without (A and B) or with (C and D) JNK inhibitor V (0.5 μM) for 30 minutes. The cells were treated with 0.1% DMSO (A) , 0.5 mM POH (B) 0.1% DMSO plus JNK inhibitor V (C) , and 0.5 mM POH plus JNK inhibitor V (D) . After 24 hours of incubation, the cells were stained with annexin V-FITC and propidium iodide and analyzed by flow cytometry. Figure 7E represents the percentage of dead cells as indicated by early apoptosis and late apoptosis or necrosis (right lower quadrant + right upper quadrant, respectively), which was calculated from the data shown in Figures 7A-D. The data were expressed as the means ± SD from at least three different experiments. ***p

    Article Snippet: Cell death assay U87 and U251 cells were pretreated for 30 minutes with JNK inhibitor V [1,3-Benzothiazol-2-yl-(2-((2-(3-pyridinyl)ethyl)amino)-4-pyrimidinyl)acetonitrile; Calbiochem], an inhibitor of JNK1/2 activation, before treatment with 0.5 mM POH and 0.5 mM POH plus 0.5 μM JNK inhibitor V. After 24 hours of incubation, the cells were suspended in annexin and propidium iodide binding buffer as specified in the TACS Annexin V-FITC apoptosis detection kit (R & D Systems).

    Techniques: Inhibition, Incubation, Staining, Flow Cytometry, Cytometry

    POH-induced apoptosis in U87 cells. U87 in the control condition (Control, A) . The cells were treated with 0.5 mM POH (POH, B) and 0.5 mM POH plus 0.5 μM JNK inhibitor V (POH + IJNK, C) . After 24 hours, the cells were immunostained for cleaved caspase-3 and the number of positive cells was analyzed (D) . Whereas POH induced cell apoptosis, the addition of the JNK inhibitor completely inhibited this effect. The addition of DMSO or JNK inhibitor V alone had no effect on cell death. Scale bar: 20 μm.*P

    Journal: Molecular Cancer

    Article Title: Na/K-ATPase as a target for anticancer drugs: studies with perillyl alcohol

    doi: 10.1186/s12943-015-0374-5

    Figure Lengend Snippet: POH-induced apoptosis in U87 cells. U87 in the control condition (Control, A) . The cells were treated with 0.5 mM POH (POH, B) and 0.5 mM POH plus 0.5 μM JNK inhibitor V (POH + IJNK, C) . After 24 hours, the cells were immunostained for cleaved caspase-3 and the number of positive cells was analyzed (D) . Whereas POH induced cell apoptosis, the addition of the JNK inhibitor completely inhibited this effect. The addition of DMSO or JNK inhibitor V alone had no effect on cell death. Scale bar: 20 μm.*P

    Article Snippet: Cell death assay U87 and U251 cells were pretreated for 30 minutes with JNK inhibitor V [1,3-Benzothiazol-2-yl-(2-((2-(3-pyridinyl)ethyl)amino)-4-pyrimidinyl)acetonitrile; Calbiochem], an inhibitor of JNK1/2 activation, before treatment with 0.5 mM POH and 0.5 mM POH plus 0.5 μM JNK inhibitor V. After 24 hours of incubation, the cells were suspended in annexin and propidium iodide binding buffer as specified in the TACS Annexin V-FITC apoptosis detection kit (R & D Systems).

    Techniques:

    The effects of JNK inhibition on the induction of cell death by POH in U87 cells. Before treatment, U87 cells were incubated without (A and B) or with (C and D) JNK inhibitor V (0.5 μM) for 30 minutes. The cells were treated with 0.1% DMSO (A), 0.5 mM POH (B) 0.1% DMSO plus JNK inhibitor V (C) , and 0.5 mM POH plus JNK inhibitor V (D) . After 24 hours of incubation, the cells were stained with annexin V-FITC and propidium iodide and analyzed by flow cytometry. Figure 6E represents the percentage of dead cells indicated by early apoptosis and late apoptosis or necrosis (right lower quadrant + right upper quadrant, respectively), which was calculated from the data shown in Figures 6A-D. The data were expressed as the means ± SD from at least three different experiments. ***p

    Journal: Molecular Cancer

    Article Title: Na/K-ATPase as a target for anticancer drugs: studies with perillyl alcohol

    doi: 10.1186/s12943-015-0374-5

    Figure Lengend Snippet: The effects of JNK inhibition on the induction of cell death by POH in U87 cells. Before treatment, U87 cells were incubated without (A and B) or with (C and D) JNK inhibitor V (0.5 μM) for 30 minutes. The cells were treated with 0.1% DMSO (A), 0.5 mM POH (B) 0.1% DMSO plus JNK inhibitor V (C) , and 0.5 mM POH plus JNK inhibitor V (D) . After 24 hours of incubation, the cells were stained with annexin V-FITC and propidium iodide and analyzed by flow cytometry. Figure 6E represents the percentage of dead cells indicated by early apoptosis and late apoptosis or necrosis (right lower quadrant + right upper quadrant, respectively), which was calculated from the data shown in Figures 6A-D. The data were expressed as the means ± SD from at least three different experiments. ***p

    Article Snippet: Cell death assay U87 and U251 cells were pretreated for 30 minutes with JNK inhibitor V [1,3-Benzothiazol-2-yl-(2-((2-(3-pyridinyl)ethyl)amino)-4-pyrimidinyl)acetonitrile; Calbiochem], an inhibitor of JNK1/2 activation, before treatment with 0.5 mM POH and 0.5 mM POH plus 0.5 μM JNK inhibitor V. After 24 hours of incubation, the cells were suspended in annexin and propidium iodide binding buffer as specified in the TACS Annexin V-FITC apoptosis detection kit (R & D Systems).

    Techniques: Inhibition, Incubation, Staining, Flow Cytometry, Cytometry