nacl  (Thermo Fisher)


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    Name:
    NaCl 5 M
    Description:
    Ambion Molecular biology grade 5 M NaCl solution is supplied in one bottle containing 500 mL The solution is certified RNase free economical and ready to use Due to the ubiquitous presence of RNases manufacturing products for use with RNA is especially challenging Ambion s nuclease free reagents and buffers are manufactured in facilities specifically designed to prevent the introduction of nucleases Highly sensitive RNase assays are performed at several different stages of the manufacturing process to ensure the highest quality These reagents are rigorously tested for contaminating nonspecific endonuclease exonuclease and RNase activity
    Catalog Number:
    am9759
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    Category:
    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher nacl
    Liquid droplet characteristics of <t>p‐tau441</t> Qualitative distribution of phosphorylation sites in p‐tau441 [pS68/69, pT153, pT175, pT181, pS184, pS199, pS202, pT205, pS210, pT212, pS214, pT217, pT231, pS235, pS262, pS324, pY310, pS316, pS396, pS404, pS422 (Mair et al )]. The charge at pH 7.4 of domains in unphosphorylated tau441 is indicated as well. Liquid–liquid phase separation (LLPS) of p‐tau441 in presence of molecular crowding (12.5% w/v Ficoll‐400). No phase separation is observed without crowding agent or in the absence of p‐tau441 protein. Liquid droplets formed by p‐tau441 in the presence of 10% (w/v) PEG were negative stained with uranyl‐acetate and visualized by transmission electron microscopy (TEM). p‐tau441 droplets are decorated with gold particles after immunogold labeling using anti‐tau antibody K9JA. . p‐tau441 droplets (in buffer with 10% PEG) exhibit glass surface wetting properties characteristics for liquids. Phase diagram of tau LLPS (p‐tau441 concentration (μM) versus PEG concentration (% w/v). In conditions modeling the intraneuronal environment (∼2 μM tau, 10% PEG, pH 7.5), p‐tau441 droplets can form at very high <t>NaCl</t> concentrations (up to 3 M NaCl) in the buffer. Guanidinium hydrochloride (GdmHCl) prevents p‐tau441 LLPS at 3 M; at this concentration, tau aggregates become visible. The chaotropic salt sodium thiocyanate (NaSCN) can inhibit LLPS with increasing concentration, whereas droplets become larger in the presence of the cosmotropic salt ammonium sulfate ((NH 4 ) 2 SO 4 ). Urea, which denatures proteins by unfolding secondary structures, prevents p‐tau441 LLPS. n = 3 per condition, determined after 3 h. Brightfield images for p‐tau441 LLPS under different salt conditions graphed in (G). The addition of 10% 1,6‐hexanediol to p‐tau441 droplets substantially reduced the amount of tau droplets formed. Data information: In (F) and (G), the phase diagrams show the average of three measurements.
    Ambion Molecular biology grade 5 M NaCl solution is supplied in one bottle containing 500 mL The solution is certified RNase free economical and ready to use Due to the ubiquitous presence of RNases manufacturing products for use with RNA is especially challenging Ambion s nuclease free reagents and buffers are manufactured in facilities specifically designed to prevent the introduction of nucleases Highly sensitive RNase assays are performed at several different stages of the manufacturing process to ensure the highest quality These reagents are rigorously tested for contaminating nonspecific endonuclease exonuclease and RNase activity
    https://www.bioz.com/result/nacl/product/Thermo Fisher
    Average 99 stars, based on 105 article reviews
    Price from $9.99 to $1999.99
    nacl - by Bioz Stars, 2021-01
    99/100 stars

    Images

    1) Product Images from "Tau protein liquid–liquid phase separation can initiate tau aggregation"

    Article Title: Tau protein liquid–liquid phase separation can initiate tau aggregation

    Journal: The EMBO Journal

    doi: 10.15252/embj.201798049

    Liquid droplet characteristics of p‐tau441 Qualitative distribution of phosphorylation sites in p‐tau441 [pS68/69, pT153, pT175, pT181, pS184, pS199, pS202, pT205, pS210, pT212, pS214, pT217, pT231, pS235, pS262, pS324, pY310, pS316, pS396, pS404, pS422 (Mair et al )]. The charge at pH 7.4 of domains in unphosphorylated tau441 is indicated as well. Liquid–liquid phase separation (LLPS) of p‐tau441 in presence of molecular crowding (12.5% w/v Ficoll‐400). No phase separation is observed without crowding agent or in the absence of p‐tau441 protein. Liquid droplets formed by p‐tau441 in the presence of 10% (w/v) PEG were negative stained with uranyl‐acetate and visualized by transmission electron microscopy (TEM). p‐tau441 droplets are decorated with gold particles after immunogold labeling using anti‐tau antibody K9JA. . p‐tau441 droplets (in buffer with 10% PEG) exhibit glass surface wetting properties characteristics for liquids. Phase diagram of tau LLPS (p‐tau441 concentration (μM) versus PEG concentration (% w/v). In conditions modeling the intraneuronal environment (∼2 μM tau, 10% PEG, pH 7.5), p‐tau441 droplets can form at very high NaCl concentrations (up to 3 M NaCl) in the buffer. Guanidinium hydrochloride (GdmHCl) prevents p‐tau441 LLPS at 3 M; at this concentration, tau aggregates become visible. The chaotropic salt sodium thiocyanate (NaSCN) can inhibit LLPS with increasing concentration, whereas droplets become larger in the presence of the cosmotropic salt ammonium sulfate ((NH 4 ) 2 SO 4 ). Urea, which denatures proteins by unfolding secondary structures, prevents p‐tau441 LLPS. n = 3 per condition, determined after 3 h. Brightfield images for p‐tau441 LLPS under different salt conditions graphed in (G). The addition of 10% 1,6‐hexanediol to p‐tau441 droplets substantially reduced the amount of tau droplets formed. Data information: In (F) and (G), the phase diagrams show the average of three measurements.
    Figure Legend Snippet: Liquid droplet characteristics of p‐tau441 Qualitative distribution of phosphorylation sites in p‐tau441 [pS68/69, pT153, pT175, pT181, pS184, pS199, pS202, pT205, pS210, pT212, pS214, pT217, pT231, pS235, pS262, pS324, pY310, pS316, pS396, pS404, pS422 (Mair et al )]. The charge at pH 7.4 of domains in unphosphorylated tau441 is indicated as well. Liquid–liquid phase separation (LLPS) of p‐tau441 in presence of molecular crowding (12.5% w/v Ficoll‐400). No phase separation is observed without crowding agent or in the absence of p‐tau441 protein. Liquid droplets formed by p‐tau441 in the presence of 10% (w/v) PEG were negative stained with uranyl‐acetate and visualized by transmission electron microscopy (TEM). p‐tau441 droplets are decorated with gold particles after immunogold labeling using anti‐tau antibody K9JA. . p‐tau441 droplets (in buffer with 10% PEG) exhibit glass surface wetting properties characteristics for liquids. Phase diagram of tau LLPS (p‐tau441 concentration (μM) versus PEG concentration (% w/v). In conditions modeling the intraneuronal environment (∼2 μM tau, 10% PEG, pH 7.5), p‐tau441 droplets can form at very high NaCl concentrations (up to 3 M NaCl) in the buffer. Guanidinium hydrochloride (GdmHCl) prevents p‐tau441 LLPS at 3 M; at this concentration, tau aggregates become visible. The chaotropic salt sodium thiocyanate (NaSCN) can inhibit LLPS with increasing concentration, whereas droplets become larger in the presence of the cosmotropic salt ammonium sulfate ((NH 4 ) 2 SO 4 ). Urea, which denatures proteins by unfolding secondary structures, prevents p‐tau441 LLPS. n = 3 per condition, determined after 3 h. Brightfield images for p‐tau441 LLPS under different salt conditions graphed in (G). The addition of 10% 1,6‐hexanediol to p‐tau441 droplets substantially reduced the amount of tau droplets formed. Data information: In (F) and (G), the phase diagrams show the average of three measurements.

    Techniques Used: Staining, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Labeling, Concentration Assay

    In vitro phase separation of tau initiated by crowding agents LLPS of p‐tau441 and p‐tau256 can also be initialized using crowding agent PEG‐8000 or a combination of PEG‐8000 with bovine serum albumin (BSA), whereas the soluble control protein GFP did not undergo LLPS in the presence of 10% PEG. We estimated the concentration of fluorescently labeled p‐tau441‐Alexa568 (10% PEG, 50 mM NaCl, 5 μM p‐tau441‐a568) in the droplets by confocal imaging ( z = 2 μm) of 30‐min‐old droplets. The measured maximum droplet fluorescence intensity was calibrated against different concentrations of p‐tau441‐Alexa568 in solution without LLPS initiation (no PEG, 50 mM NaCl). Cross‐sectional profile along the white arrow visualizes the intensity levels of droplets. The mean of the detected p‐tau441‐a568 concentration was 34.3 ± 6.4 μM in the droplets and 3.6 ± 0.3 μM in the solution phase. This value might be underestimating the actual tau concentration in the droplets because fluorophore:fluorophore quenching and other artifact resulting from the highly viscous and crowded environment in the droplets are not considered. Absorbance spectra of free and p‐tau441 bound Alexa568 shows differences in the intensity but no shift in the wavelengths. The intensity of free Alexa568 dye imaged at 533 nm in confocal microscopy did not change with the amount of PEG in solution. By confocal imaging, the crowding agent dextran remains excluded from p‐tau441 droplets initiated by adding 9% dextran‐70 kDa and 1% of fluorescent dextrans of different molecular weights. The addition of methylene blue, viscous aqua, and ThioS, all dyes with affinity to hydrophobic protein areas, to freshly prepared p‐tau441 droplets reveals the co‐partitioning and retention of these dyes in the droplets. The fluorescence may be further enhanced by inhibition of free rotation of the dyes due to the higher droplet viscosity. Data information: In right graphs of (B, C), data are represented as mean ± s.d.
    Figure Legend Snippet: In vitro phase separation of tau initiated by crowding agents LLPS of p‐tau441 and p‐tau256 can also be initialized using crowding agent PEG‐8000 or a combination of PEG‐8000 with bovine serum albumin (BSA), whereas the soluble control protein GFP did not undergo LLPS in the presence of 10% PEG. We estimated the concentration of fluorescently labeled p‐tau441‐Alexa568 (10% PEG, 50 mM NaCl, 5 μM p‐tau441‐a568) in the droplets by confocal imaging ( z = 2 μm) of 30‐min‐old droplets. The measured maximum droplet fluorescence intensity was calibrated against different concentrations of p‐tau441‐Alexa568 in solution without LLPS initiation (no PEG, 50 mM NaCl). Cross‐sectional profile along the white arrow visualizes the intensity levels of droplets. The mean of the detected p‐tau441‐a568 concentration was 34.3 ± 6.4 μM in the droplets and 3.6 ± 0.3 μM in the solution phase. This value might be underestimating the actual tau concentration in the droplets because fluorophore:fluorophore quenching and other artifact resulting from the highly viscous and crowded environment in the droplets are not considered. Absorbance spectra of free and p‐tau441 bound Alexa568 shows differences in the intensity but no shift in the wavelengths. The intensity of free Alexa568 dye imaged at 533 nm in confocal microscopy did not change with the amount of PEG in solution. By confocal imaging, the crowding agent dextran remains excluded from p‐tau441 droplets initiated by adding 9% dextran‐70 kDa and 1% of fluorescent dextrans of different molecular weights. The addition of methylene blue, viscous aqua, and ThioS, all dyes with affinity to hydrophobic protein areas, to freshly prepared p‐tau441 droplets reveals the co‐partitioning and retention of these dyes in the droplets. The fluorescence may be further enhanced by inhibition of free rotation of the dyes due to the higher droplet viscosity. Data information: In right graphs of (B, C), data are represented as mean ± s.d.

    Techniques Used: In Vitro, Concentration Assay, Labeling, Imaging, Fluorescence, Confocal Microscopy, Inhibition

    2) Product Images from "GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana"

    Article Title: GmWRKY16 Enhances Drought and Salt Tolerance Through an ABA-Mediated Pathway in Arabidopsis thaliana

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.01979

    Expression patterns of stress/ABA responsive genes regulated by GmWRKY16 . (A) Expression patterns of genes responsive to salt stress. (B) Expression patterns of ABA and/or stress-responsive genes under drought stress. (C) Expression patterns of ABA-responsive genes under drought stress. Arabidopsis seeds of WT and GmWRKY16 transgenic line #12 of T 3 generation were sown in mixed soil (vermiculite and flower nutrient soil, 1:1) and cultured in the chamber room. The 2-week-old seedlings were subjected to drought treatment (withholding water) and 200 mM NaCl treatment for 10 days. The samples from the aerial parts of Arabidopsis plants were taken for total RNA extraction. The relative expression of drought- and/or salt stress-responsive genes was quantified by qRT-PCR using ACT3 as the reference gene to normalize the data ( ∗∗ P = 0.01). The 2 -ΔΔCt method was used to evaluate the quantitative variation between the examined replicates ( Lü et al., 2015 ). The details for the specific primers of the GmWRKY16 , ACT3 and stress-responsive genes are listed in Supplementary Table S1 .
    Figure Legend Snippet: Expression patterns of stress/ABA responsive genes regulated by GmWRKY16 . (A) Expression patterns of genes responsive to salt stress. (B) Expression patterns of ABA and/or stress-responsive genes under drought stress. (C) Expression patterns of ABA-responsive genes under drought stress. Arabidopsis seeds of WT and GmWRKY16 transgenic line #12 of T 3 generation were sown in mixed soil (vermiculite and flower nutrient soil, 1:1) and cultured in the chamber room. The 2-week-old seedlings were subjected to drought treatment (withholding water) and 200 mM NaCl treatment for 10 days. The samples from the aerial parts of Arabidopsis plants were taken for total RNA extraction. The relative expression of drought- and/or salt stress-responsive genes was quantified by qRT-PCR using ACT3 as the reference gene to normalize the data ( ∗∗ P = 0.01). The 2 -ΔΔCt method was used to evaluate the quantitative variation between the examined replicates ( Lü et al., 2015 ). The details for the specific primers of the GmWRKY16 , ACT3 and stress-responsive genes are listed in Supplementary Table S1 .

    Techniques Used: Expressing, Transgenic Assay, Cell Culture, RNA Extraction, Quantitative RT-PCR

    3) Product Images from "Release of Periplasmic Proteins of Brucella suis upon Acidic Shock Involves the Outer Membrane Protein Omp25 "

    Article Title: Release of Periplasmic Proteins of Brucella suis upon Acidic Shock Involves the Outer Membrane Protein Omp25

    Journal: Infection and Immunity

    doi: 10.1128/IAI.72.10.5693-5703.2004

    Release of proteins into bacterial supernatant increases in acidic acetate buffer (pH 4). Wild-type B. suis 1330 cells were incubated for 150 min in various media before the bacteria were centrifuged and the different supernatants were concentrated 1,000-fold. Brucella proteins from the lysate or the concentrated supernatants were separated by SDS-PAGE and stained with Coomassie blue (A) or blotted and immunostained with an anti-Omp25 antibody (B). Lanes: 1, bacterial lysate; 2, supernatants composed of ammonium acetate containing 120 mM NaCl, pH 4; 3, PBS, pH 7; 4, RPMI, pH 7.5; Markers, molecular mass markers.
    Figure Legend Snippet: Release of proteins into bacterial supernatant increases in acidic acetate buffer (pH 4). Wild-type B. suis 1330 cells were incubated for 150 min in various media before the bacteria were centrifuged and the different supernatants were concentrated 1,000-fold. Brucella proteins from the lysate or the concentrated supernatants were separated by SDS-PAGE and stained with Coomassie blue (A) or blotted and immunostained with an anti-Omp25 antibody (B). Lanes: 1, bacterial lysate; 2, supernatants composed of ammonium acetate containing 120 mM NaCl, pH 4; 3, PBS, pH 7; 4, RPMI, pH 7.5; Markers, molecular mass markers.

    Techniques Used: Incubation, SDS Page, Staining

    4) Product Images from "A monovalent ion in the DNA binding interface of the eukaryotic junction-resolving enzyme GEN1"

    Article Title: A monovalent ion in the DNA binding interface of the eukaryotic junction-resolving enzyme GEN1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky863

    Cleavage of branched DNA substrates by C tGEN1 as a function of the monovalent metal ion present. Substrates radioactively [5′- 32 P]-labeled on the x strand were incubated with Ct GEN1 in buffer containing 10 mM cacodylate (pH 6.5), 1 mM MgCl 2, 50 mM NaCl or KCl, 0.1% BSA for 3 min at 37°C. The products separated by polyacrylamide gel electrophoresis followed by phosphorimaging. ( A ) Denaturing gel electrophoresis. The major product of cleavage is arrowed. Note that each substrate is cleaved at the same position. The scheme on the right shows the sequence of the core of the junction, and the nomenclature of the arms and strands. The position of the radioactive label is indicated and the cleavage sites are arrowed. The major cleavages are made in the B and R arms, 1 nt 3′ to the point of strand exchange on the h and x strands. In this experiment the x strand is radioactively [5′- 32 P]-labeled, and thus only cleavage in this strand is detected. ( B ) Native gel electrophoresis. The products of Ct GEN1 cleavage are arrowed, with the relevant tracks indicated; e.g. the arrow labeled 1–3 indicates the product of four-way junction cleavage. In A and B tracks 1–3 contain the four-way junction (4H), tracks 4–6 contain the nicked three-way junction, tracks 7–9 contain the three-way junction (3H) and tracks 10–12 contain the splayed helix junction. For each is shown no cleavage with Ct GEN1 i.e. intact junction, tracks 1, 4, 7 and 10, cleavage with Ct GEN1 in the presence of Na + ions, tracks 2, 5, 8 and 11, and cleavage with Ct GEN1 in the presence of K + ions, tracks 3, 6, 9 and 12. ( C ) Progress curve for cleavage of a nicked three-way junction. ( D ) Progress curve for cleavage of a splayed helix junction. In both C and D cleavage in K + and Na + .
    Figure Legend Snippet: Cleavage of branched DNA substrates by C tGEN1 as a function of the monovalent metal ion present. Substrates radioactively [5′- 32 P]-labeled on the x strand were incubated with Ct GEN1 in buffer containing 10 mM cacodylate (pH 6.5), 1 mM MgCl 2, 50 mM NaCl or KCl, 0.1% BSA for 3 min at 37°C. The products separated by polyacrylamide gel electrophoresis followed by phosphorimaging. ( A ) Denaturing gel electrophoresis. The major product of cleavage is arrowed. Note that each substrate is cleaved at the same position. The scheme on the right shows the sequence of the core of the junction, and the nomenclature of the arms and strands. The position of the radioactive label is indicated and the cleavage sites are arrowed. The major cleavages are made in the B and R arms, 1 nt 3′ to the point of strand exchange on the h and x strands. In this experiment the x strand is radioactively [5′- 32 P]-labeled, and thus only cleavage in this strand is detected. ( B ) Native gel electrophoresis. The products of Ct GEN1 cleavage are arrowed, with the relevant tracks indicated; e.g. the arrow labeled 1–3 indicates the product of four-way junction cleavage. In A and B tracks 1–3 contain the four-way junction (4H), tracks 4–6 contain the nicked three-way junction, tracks 7–9 contain the three-way junction (3H) and tracks 10–12 contain the splayed helix junction. For each is shown no cleavage with Ct GEN1 i.e. intact junction, tracks 1, 4, 7 and 10, cleavage with Ct GEN1 in the presence of Na + ions, tracks 2, 5, 8 and 11, and cleavage with Ct GEN1 in the presence of K + ions, tracks 3, 6, 9 and 12. ( C ) Progress curve for cleavage of a nicked three-way junction. ( D ) Progress curve for cleavage of a splayed helix junction. In both C and D cleavage in K + and Na + .

    Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Nucleic Acid Electrophoresis, Sequencing

    5) Product Images from "Novel Hepatitis B Virus Capsid-Targeting Antiviral that Aggregates Core Particles and Inhibits Nuclear Entry of Viral Cores"

    Article Title: Novel Hepatitis B Virus Capsid-Targeting Antiviral that Aggregates Core Particles and Inhibits Nuclear Entry of Viral Cores

    Journal: ACS infectious diseases

    doi: 10.1021/acsinfecdis.8b00235

    Development and validation of thermal shift assay for Cp binders. (A) Schematic of thermal shift assay (TSA). (B) TSA was performed under non-assembly (50 mM sodium bicarbonate, pH 9.6, no NaCl, no pre-incubation) and full assembly (50 mM HEPES, pH 7.5, 0.5 M NaCl, 30 min pre-incubation at 37°C) conditions for Cp (7.5 μM dimer). (C-D) TSA was performed for Cp (7.5 μM dimer) in the presence or absence of 20 μM (C) Bay 38-7690 or (D) DVR - 56
    Figure Legend Snippet: Development and validation of thermal shift assay for Cp binders. (A) Schematic of thermal shift assay (TSA). (B) TSA was performed under non-assembly (50 mM sodium bicarbonate, pH 9.6, no NaCl, no pre-incubation) and full assembly (50 mM HEPES, pH 7.5, 0.5 M NaCl, 30 min pre-incubation at 37°C) conditions for Cp (7.5 μM dimer). (C-D) TSA was performed for Cp (7.5 μM dimer) in the presence or absence of 20 μM (C) Bay 38-7690 or (D) DVR - 56

    Techniques Used: Thermal Shift Assay, Incubation

    6) Product Images from "Proteolysis of Iron Oxide-Associated Bovine Serum Albumin"

    Article Title: Proteolysis of Iron Oxide-Associated Bovine Serum Albumin

    Journal: Environmental Science & Technology

    doi: 10.1021/acs.est.0c00860

    Proteolysis of goethite-associated BSA at 1.4 mg BSA m –2 , with or without coadsorbed phosphate, determined from either batch or IR experiments. The proteolysis was measured as the decrease of the intensity of the BSA peak in SEC ( cf. Figure 2 B,C) and the amide II band in the batch and IR experiment, respectively. All values were normalized against the initial value ( t = 0). The proteolysis of aqueous BSA at a concentration of 100 mg L –1 is shown for comparison. All experiments were performed at pH 4.0 in 0.01 M NaCl, and the protease concentration was 5 mg L –1 . Error bars represent standard deviations ( n = 3 for goethite-associated BSA and n = 2 for aqueous BSA).
    Figure Legend Snippet: Proteolysis of goethite-associated BSA at 1.4 mg BSA m –2 , with or without coadsorbed phosphate, determined from either batch or IR experiments. The proteolysis was measured as the decrease of the intensity of the BSA peak in SEC ( cf. Figure 2 B,C) and the amide II band in the batch and IR experiment, respectively. All values were normalized against the initial value ( t = 0). The proteolysis of aqueous BSA at a concentration of 100 mg L –1 is shown for comparison. All experiments were performed at pH 4.0 in 0.01 M NaCl, and the protease concentration was 5 mg L –1 . Error bars represent standard deviations ( n = 3 for goethite-associated BSA and n = 2 for aqueous BSA).

    Techniques Used: Concentration Assay

    MCR analysis of IR spectral data sets of goethite-associated BSA during 20 h proteolysis reaction at 0.7 (low coverage, A) and 1.4 mg BSA m –2 (high coverage, B) and at pH 4.0 in 0.01 M NaCl. The spectra of components 1 (C1) and 2 (C2) are presented as dotted and solid lines, respectively, and the relative contribution of C1 and C2 during the proteolysis is shown as insets. Note that the spectra of C1 and C2 are presented as the unmodified output from the MCR analysis, that is, they have not been scaled separately after the analysis. The numbers in the plain and bold text indicate the main peak positions of C1 and C2, respectively. The first time point was collected after ca. 1 min.
    Figure Legend Snippet: MCR analysis of IR spectral data sets of goethite-associated BSA during 20 h proteolysis reaction at 0.7 (low coverage, A) and 1.4 mg BSA m –2 (high coverage, B) and at pH 4.0 in 0.01 M NaCl. The spectra of components 1 (C1) and 2 (C2) are presented as dotted and solid lines, respectively, and the relative contribution of C1 and C2 during the proteolysis is shown as insets. Note that the spectra of C1 and C2 are presented as the unmodified output from the MCR analysis, that is, they have not been scaled separately after the analysis. The numbers in the plain and bold text indicate the main peak positions of C1 and C2, respectively. The first time point was collected after ca. 1 min.

    Techniques Used:

    Area-normalized SEC chromatograms of 100 mg L –1 BSA in aqueous solution (A) and the phosphate-desorbed fraction of ferrihydrite-associated BSA (B) and goethite-associated BSA (C), before ( T 0) and after 3 h proteolysis reaction ( T 3h). The experiments were performed at a BSA surface coverage of 1.4 mg m –2 (corresponding to total BSA concentrations of 200 and 100 mg L –1 in the ferrihydrite and goethite systems, respectively) at pH 4.0 in 0.01 M NaCl and at a protease concentration of 10 mg L –1 . The insets show the difference between the chromatograms at T 3h and T 0. The horizontal dotted lines in the insets indicate the 0-level. The shaded bands represent standard deviations ( n = 2). The molecular masses (in Da) of peptide standards are represented by the vertical dotted lines.
    Figure Legend Snippet: Area-normalized SEC chromatograms of 100 mg L –1 BSA in aqueous solution (A) and the phosphate-desorbed fraction of ferrihydrite-associated BSA (B) and goethite-associated BSA (C), before ( T 0) and after 3 h proteolysis reaction ( T 3h). The experiments were performed at a BSA surface coverage of 1.4 mg m –2 (corresponding to total BSA concentrations of 200 and 100 mg L –1 in the ferrihydrite and goethite systems, respectively) at pH 4.0 in 0.01 M NaCl and at a protease concentration of 10 mg L –1 . The insets show the difference between the chromatograms at T 3h and T 0. The horizontal dotted lines in the insets indicate the 0-level. The shaded bands represent standard deviations ( n = 2). The molecular masses (in Da) of peptide standards are represented by the vertical dotted lines.

    Techniques Used: Concentration Assay

    Adsorption isotherms of BSA on ferrihydrite (A) and goethite (B). The experiments were conducted at pH 4.0 in 0.01 M NaCl for 24 h. The data are compared with a 1:1 line (dotted). Arrows indicate the total BSA concentrations added in the proteolysis experiments.
    Figure Legend Snippet: Adsorption isotherms of BSA on ferrihydrite (A) and goethite (B). The experiments were conducted at pH 4.0 in 0.01 M NaCl for 24 h. The data are compared with a 1:1 line (dotted). Arrows indicate the total BSA concentrations added in the proteolysis experiments.

    Techniques Used: Adsorption

    Difference in SEC chromatograms of the phosphate-desorbed fraction of ferrihydrite-associated BSA (A) and goethite-associated BSA (B) in the presence of coadsorbed phosphate after 3 h proteolysis reaction. The experiments were performed at 0.7 mg BSA m –2 and pH 4.0 in 0.01 M NaCl, and the protease concentration was 10 mg L –1 . The total added phosphate concentrations were 1.0, 2.0, and 5.0 μmol m –2 , which are indicated by numbers in the figure legends. The difference chromatograms shown are obtained by subtraction with a control without phosphate addition. The horizontal dotted lines indicate the 0-level. The shaded bands represent standard deviations ( n = 2). The arrows indicate the direction of change, and the vertical dotted lines represent the molecular masses (in Da) of peptide standards.
    Figure Legend Snippet: Difference in SEC chromatograms of the phosphate-desorbed fraction of ferrihydrite-associated BSA (A) and goethite-associated BSA (B) in the presence of coadsorbed phosphate after 3 h proteolysis reaction. The experiments were performed at 0.7 mg BSA m –2 and pH 4.0 in 0.01 M NaCl, and the protease concentration was 10 mg L –1 . The total added phosphate concentrations were 1.0, 2.0, and 5.0 μmol m –2 , which are indicated by numbers in the figure legends. The difference chromatograms shown are obtained by subtraction with a control without phosphate addition. The horizontal dotted lines indicate the 0-level. The shaded bands represent standard deviations ( n = 2). The arrows indicate the direction of change, and the vertical dotted lines represent the molecular masses (in Da) of peptide standards.

    Techniques Used: Concentration Assay

    7) Product Images from "High NaCl-induced inhibition of PTG contributes to activation of NFAT5 through attenuation of the negative effect of SHP-1"

    Article Title: High NaCl-induced inhibition of PTG contributes to activation of NFAT5 through attenuation of the negative effect of SHP-1

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00218.2013

    A : PTG-V5 is physically associated with SHP-1 but not SHP-2, and the association is not affected by high NaCl. HEK293 cells were co-transfected at 290 mosM with plasmids coding for PTG-V5 and with SHP-1-HA or SHP-2-myc for 48 h. Then, osmolality was increased
    Figure Legend Snippet: A : PTG-V5 is physically associated with SHP-1 but not SHP-2, and the association is not affected by high NaCl. HEK293 cells were co-transfected at 290 mosM with plasmids coding for PTG-V5 and with SHP-1-HA or SHP-2-myc for 48 h. Then, osmolality was increased

    Techniques Used: Transfection

    8) Product Images from "Structural model of ubiquitin transfer onto an artificial RING finger as an E3 ligase"

    Article Title: Structural model of ubiquitin transfer onto an artificial RING finger as an E3 ligase

    Journal: Scientific Reports

    doi: 10.1038/srep06574

    CD spectra of the artificial WSTF PHD_EL5 RING finger and its five mutants. Spectra of 25 μM samples were collected in 20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, and 50 μM ZnCl 2 at room temperature. (1) K4R, (2) K8R, (3) K9R, (4) K14R, and (5) K23R are denoted by solid lines, and the dotted line displays the wild-type.
    Figure Legend Snippet: CD spectra of the artificial WSTF PHD_EL5 RING finger and its five mutants. Spectra of 25 μM samples were collected in 20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, and 50 μM ZnCl 2 at room temperature. (1) K4R, (2) K8R, (3) K9R, (4) K14R, and (5) K23R are denoted by solid lines, and the dotted line displays the wild-type.

    Techniques Used:

    9) Product Images from "The RNA chaperone activity of the Trypanosoma brucei editosome raises the dynamic of bound pre-mRNAs"

    Article Title: The RNA chaperone activity of the Trypanosoma brucei editosome raises the dynamic of bound pre-mRNAs

    Journal: Scientific Reports

    doi: 10.1038/srep19309

    Experimental verification of GQ-folds - thermal difference (TD) spectra. Normalized double-difference TD-spectra of the A6- and ND3-transcript in the presence of KCl (red), NaCl (blue) and LiCl (green). Signature peaks of GQ-folds are marked by arrows 23 . Additional differences are indicated by arrowheads. AU: arbitrary unit. Peak intensities decrease in the presence of GQ-destabilizing Na + - and Li + -ions.
    Figure Legend Snippet: Experimental verification of GQ-folds - thermal difference (TD) spectra. Normalized double-difference TD-spectra of the A6- and ND3-transcript in the presence of KCl (red), NaCl (blue) and LiCl (green). Signature peaks of GQ-folds are marked by arrows 23 . Additional differences are indicated by arrowheads. AU: arbitrary unit. Peak intensities decrease in the presence of GQ-destabilizing Na + - and Li + -ions.

    Techniques Used:

    10) Product Images from "Calcineurin and Calcium Channel CchA Coordinate the Salt Stress Response by Regulating Cytoplasmic Ca2+ Homeostasis in Aspergillus nidulans"

    Article Title: Calcineurin and Calcium Channel CchA Coordinate the Salt Stress Response by Regulating Cytoplasmic Ca2+ Homeostasis in Aspergillus nidulans

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00330-16

    The hyphal growth defects of the Δ cnaA or Δ cnaB strains could be suppressed by the dysfunction of CchA under salt stress. (A) Colony morphology of the Δ cnaA , Δ cnaA cchA re , Δ cnaB , and Δ cnaB Δ cchA strains and the reference strain. The conidia were spotted on solid MMPDRUU and MMPDRUU supplemented with 800 mM NaCl, 600 mM KCl, or 1 M sorbitol, respectively, at 37°C for 2.5 days. (B) Graphic representation of radial growth rates of the Δ cnaA and Δ cnaA cchA re strains and the reference strain. The values are means ± standard deviations (SD) from three independent experiments. (C) Differential interference contrast images of hyphae grown on MMPDRUU in the presence or absence of salt stress at 37°C for 16 h. Bar, 10 μm. (D) Analysis of branching frequencies in hyphal filaments of the Δ cnaA and Δ cnaA cchA re strains and the reference strain. The values are means ± SD from three independent experiments.
    Figure Legend Snippet: The hyphal growth defects of the Δ cnaA or Δ cnaB strains could be suppressed by the dysfunction of CchA under salt stress. (A) Colony morphology of the Δ cnaA , Δ cnaA cchA re , Δ cnaB , and Δ cnaB Δ cchA strains and the reference strain. The conidia were spotted on solid MMPDRUU and MMPDRUU supplemented with 800 mM NaCl, 600 mM KCl, or 1 M sorbitol, respectively, at 37°C for 2.5 days. (B) Graphic representation of radial growth rates of the Δ cnaA and Δ cnaA cchA re strains and the reference strain. The values are means ± standard deviations (SD) from three independent experiments. (C) Differential interference contrast images of hyphae grown on MMPDRUU in the presence or absence of salt stress at 37°C for 16 h. Bar, 10 μm. (D) Analysis of branching frequencies in hyphal filaments of the Δ cnaA and Δ cnaA cchA re strains and the reference strain. The values are means ± SD from three independent experiments.

    Techniques Used:

    Colony morphologies of the Δ yvcA , Δ cnaA , and Δ cnaA Δ yvcA strains grown on MMPDRUU in the presence or absence of 800 mM NaCl or 800 mM NaCl plus 3 mM EGTA at 37°C for 2.5 days.
    Figure Legend Snippet: Colony morphologies of the Δ yvcA , Δ cnaA , and Δ cnaA Δ yvcA strains grown on MMPDRUU in the presence or absence of 800 mM NaCl or 800 mM NaCl plus 3 mM EGTA at 37°C for 2.5 days.

    Techniques Used:

    Expression analysis of Ca 2+ -signaling-related and salt-stress-induced genes in response to salt stress by quantitative PCR. (A) Fold changes in mRNA levels, including vcxA , yvcA , pmrA , and pmcA , after incubation with MMPDRUU with addition of 800 mM NaCl compared to results with MMPDRUU alone using real-time RT-PCR. (B) Fold changes in mRNA levels, including enaA , nhaA , and trkA , after incubation with MMPDRUU with addition of 800 mM NaCl compared to results with MMPDRUU alone using real-time RT-PCR. Data representing the indicated strains' mRNA levels from salt stress pretreatment were normalized to the non-salt-pretreatment condition. The error bars indicate the standard deviations from three independent replicates.
    Figure Legend Snippet: Expression analysis of Ca 2+ -signaling-related and salt-stress-induced genes in response to salt stress by quantitative PCR. (A) Fold changes in mRNA levels, including vcxA , yvcA , pmrA , and pmcA , after incubation with MMPDRUU with addition of 800 mM NaCl compared to results with MMPDRUU alone using real-time RT-PCR. (B) Fold changes in mRNA levels, including enaA , nhaA , and trkA , after incubation with MMPDRUU with addition of 800 mM NaCl compared to results with MMPDRUU alone using real-time RT-PCR. Data representing the indicated strains' mRNA levels from salt stress pretreatment were normalized to the non-salt-pretreatment condition. The error bars indicate the standard deviations from three independent replicates.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Incubation, Quantitative RT-PCR

    11) Product Images from "Monitoring Intracellular pH change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells"

    Article Title: Monitoring Intracellular pH change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-4939-3813-1_9

    pH response of purified pHlash protein in vitro (A) SDS-PAGE gel of purified His-tagged pHlash protein stained with Coomassie Blue dye. Leftmost lane is molecular weight standards with KDa indicated, while the other lanes are the purified pHlash protein loaded at 0.2, 1, and 2 μg per lane. (B ) Construct of the pHlash fusion protein. Rluc8 was linked to cpVenus by the sequence Ala-Glu-Leu. (C ) Raw data (not normalized) of luminescence emission spectra of purified pHlash protein with 10 μM native coelenterazine at different pH values (pH 5.4-9.0) in 50 mM BIS-Tris-propane, 100 mM KCl, and 100 mM NaCl. (D) Normalized luminescence emission spectra of pHlash measured as in panel C. Luminescence intensity was normalized to the peak at 475 nm. (E) The BRET ratio (luminescence at 525nm:475 nm) as a function of pH is shown for pHlash in vitro . Error bars are +/- S.D., but in most cases the error bars are so small that they are obscured by the symbols (n = 3).
    Figure Legend Snippet: pH response of purified pHlash protein in vitro (A) SDS-PAGE gel of purified His-tagged pHlash protein stained with Coomassie Blue dye. Leftmost lane is molecular weight standards with KDa indicated, while the other lanes are the purified pHlash protein loaded at 0.2, 1, and 2 μg per lane. (B ) Construct of the pHlash fusion protein. Rluc8 was linked to cpVenus by the sequence Ala-Glu-Leu. (C ) Raw data (not normalized) of luminescence emission spectra of purified pHlash protein with 10 μM native coelenterazine at different pH values (pH 5.4-9.0) in 50 mM BIS-Tris-propane, 100 mM KCl, and 100 mM NaCl. (D) Normalized luminescence emission spectra of pHlash measured as in panel C. Luminescence intensity was normalized to the peak at 475 nm. (E) The BRET ratio (luminescence at 525nm:475 nm) as a function of pH is shown for pHlash in vitro . Error bars are +/- S.D., but in most cases the error bars are so small that they are obscured by the symbols (n = 3).

    Techniques Used: Purification, In Vitro, SDS Page, Staining, Molecular Weight, Construct, Sequencing, Bioluminescence Resonance Energy Transfer

    12) Product Images from "Synthesis of adenine dinucleosides SAM analogs as specific inhibitors of SARS-CoV nsp14 RNA cap guanine-N7-methyltransferase"

    Article Title: Synthesis of adenine dinucleosides SAM analogs as specific inhibitors of SARS-CoV nsp14 RNA cap guanine-N7-methyltransferase

    Journal: European Journal of Medicinal Chemistry

    doi: 10.1016/j.ejmech.2020.112557

    Thermal shifts (ΔTm) of SARS-CoV nsp14 in the absence or presence of SAM, sinefungin and 9 dinucleoside inhibitors 6 and 9 – 16 . Thermal stability of SARS-CoV nsp14 upon ligand binding was monitored by differential scanning fluorimetry. Assays were carried out in reaction mixture [20 mM HEPES (pH 7.5), 150 mM NaCl, 1x SYPRO orange dye] in the presence of 5 μM SARS-CoV nsp14 protein and 1 mM compound previously dissolved in 100% DMSO. The bars and error bars correspond to the mean values from three independent measurements and their s.d.’s, respectively.
    Figure Legend Snippet: Thermal shifts (ΔTm) of SARS-CoV nsp14 in the absence or presence of SAM, sinefungin and 9 dinucleoside inhibitors 6 and 9 – 16 . Thermal stability of SARS-CoV nsp14 upon ligand binding was monitored by differential scanning fluorimetry. Assays were carried out in reaction mixture [20 mM HEPES (pH 7.5), 150 mM NaCl, 1x SYPRO orange dye] in the presence of 5 μM SARS-CoV nsp14 protein and 1 mM compound previously dissolved in 100% DMSO. The bars and error bars correspond to the mean values from three independent measurements and their s.d.’s, respectively.

    Techniques Used: Ligand Binding Assay

    13) Product Images from "Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1)"

    Article Title: Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1)

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1038/mtm.2015.58

    Effects of chromatography-based purification of recombinant adeno-associated virus serotype 1 (rAAV1). ( a ) rAAV1 eluted from the five Mustang QXTs (anion-exchange column) by stepwise NaCl gradient elution was analyzed by 5–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel (Oriole staining). The black square indicates the fractions loaded on Superdex 200 10/300 GL at next gel-filtration chromatography step. The white triangle indicates the 200 kDa protein impurities. Lane 1: after dialysis; Lanes 2–4: eluted by 100 mmol/l NaCl; Lanes 5–7: eluted by 120 mmol/l NaCl; Lanes 8–10: eluted by 130 mmol/l NaCl; Lanes 11–13: eluted by 140 mmol/l NaCl; Lanes 14–16: eluted by 200 mmol/l NaCl; M: protein size marker. ( b,c ) The samples were subsequently gel-filtrated by the Superdex 200 10/300 GL column and elution fractions were analyzed by 5–20% gradient SDS–PAGE (Oriole staining). (b) Elution pattern of Superdex 200 10/300 GL. The y-axis: 280 nm absorbance; x-axis: fraction number. The black triangle indicates the peak fractions of rAAV1 (corresponding to the black square on (c)) and the gray arrowhead indicates ~65 kDa protein impurities. (c) Elution fractions analyzed by 5–20% gradient SDS–PAGE gel (Oriole staining). Lane 1: after dialysis; Lanes 2–11: Fr 13–22; Lane 12: Fr 27; M: protein size marker. The gray arrowhead shows the same as (b). ( d–f ) Peak fractions (the black square on ( c )) were collected and concentrated by Ultracel 30 K to obtain the final product. ( d ) The final product of rAAV1 was analyzed by 5–20% gradient SDS–PAGE gel (Oriole staining), ( e ) western blotting, and ( f ) electron microscopy (negative staining). M: protein size marker. The three rAAV1 capsid proteins, VP1 (81.4 kDa), VP2 (66.2 kDa), and VP3 (59.6 kDa), were represented.
    Figure Legend Snippet: Effects of chromatography-based purification of recombinant adeno-associated virus serotype 1 (rAAV1). ( a ) rAAV1 eluted from the five Mustang QXTs (anion-exchange column) by stepwise NaCl gradient elution was analyzed by 5–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel (Oriole staining). The black square indicates the fractions loaded on Superdex 200 10/300 GL at next gel-filtration chromatography step. The white triangle indicates the 200 kDa protein impurities. Lane 1: after dialysis; Lanes 2–4: eluted by 100 mmol/l NaCl; Lanes 5–7: eluted by 120 mmol/l NaCl; Lanes 8–10: eluted by 130 mmol/l NaCl; Lanes 11–13: eluted by 140 mmol/l NaCl; Lanes 14–16: eluted by 200 mmol/l NaCl; M: protein size marker. ( b,c ) The samples were subsequently gel-filtrated by the Superdex 200 10/300 GL column and elution fractions were analyzed by 5–20% gradient SDS–PAGE (Oriole staining). (b) Elution pattern of Superdex 200 10/300 GL. The y-axis: 280 nm absorbance; x-axis: fraction number. The black triangle indicates the peak fractions of rAAV1 (corresponding to the black square on (c)) and the gray arrowhead indicates ~65 kDa protein impurities. (c) Elution fractions analyzed by 5–20% gradient SDS–PAGE gel (Oriole staining). Lane 1: after dialysis; Lanes 2–11: Fr 13–22; Lane 12: Fr 27; M: protein size marker. The gray arrowhead shows the same as (b). ( d–f ) Peak fractions (the black square on ( c )) were collected and concentrated by Ultracel 30 K to obtain the final product. ( d ) The final product of rAAV1 was analyzed by 5–20% gradient SDS–PAGE gel (Oriole staining), ( e ) western blotting, and ( f ) electron microscopy (negative staining). M: protein size marker. The three rAAV1 capsid proteins, VP1 (81.4 kDa), VP2 (66.2 kDa), and VP3 (59.6 kDa), were represented.

    Techniques Used: Chromatography, Purification, Recombinant, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Filtration, Marker, Western Blot, Electron Microscopy, Negative Staining

    Purification with a buffer containing 50 mmol/l NaCl. ( a ) Recombinant adeno-associated virus serotype 1 (rAAV1) purified by ion-exchange column with a buffer containing 50 mmol/l NaCl was analyzed by 5–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Oriole staining) and ( b ) western blotting. M: protein size marker. The 200 kDa protein impurities were still present (white triangle) and the purity was low. The three rAAV1 capsid proteins, VP1 (81.4 kDa), VP2 (66.2 kDa), and VP3 (59.6 kDa), were represented.
    Figure Legend Snippet: Purification with a buffer containing 50 mmol/l NaCl. ( a ) Recombinant adeno-associated virus serotype 1 (rAAV1) purified by ion-exchange column with a buffer containing 50 mmol/l NaCl was analyzed by 5–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Oriole staining) and ( b ) western blotting. M: protein size marker. The 200 kDa protein impurities were still present (white triangle) and the purity was low. The three rAAV1 capsid proteins, VP1 (81.4 kDa), VP2 (66.2 kDa), and VP3 (59.6 kDa), were represented.

    Techniques Used: Purification, Recombinant, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Western Blot, Marker

    14) Product Images from "Dual role of SND1 facilitates efficient communication between abiotic stress signalling and normal growth in Arabidopsis"

    Article Title: Dual role of SND1 facilitates efficient communication between abiotic stress signalling and normal growth in Arabidopsis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-28413-x

    Relative expression of the SND1 transcript in plants subjected to different hormone treatments or abiotic stresses. The relative expression of SND1 transcript in Col-0 was determined by the qRT-PCR. Eight-day-old seedlings were used to extract mRNA following the treatment with 10 µM abscisic acid (ABA), 10 µM indole-3-acetic acid (IAA), 10 µM jasmonic acid (JA), 10 µM salicylic acid (SA), 300 mM sucrose (Suc), 400 mM mannitol (Man), and 300 mM NaCl for 6 h. The error bars indicate the standard error (SE) of three replicates. The values with different letters were significantly different from that of WT plants (P
    Figure Legend Snippet: Relative expression of the SND1 transcript in plants subjected to different hormone treatments or abiotic stresses. The relative expression of SND1 transcript in Col-0 was determined by the qRT-PCR. Eight-day-old seedlings were used to extract mRNA following the treatment with 10 µM abscisic acid (ABA), 10 µM indole-3-acetic acid (IAA), 10 µM jasmonic acid (JA), 10 µM salicylic acid (SA), 300 mM sucrose (Suc), 400 mM mannitol (Man), and 300 mM NaCl for 6 h. The error bars indicate the standard error (SE) of three replicates. The values with different letters were significantly different from that of WT plants (P

    Techniques Used: Expressing, Quantitative RT-PCR

    15) Product Images from "Mutational and Metal Binding Analysis of the Endonuclease Domain of the Influenza Virus Polymerase PA Subunit ▿"

    Article Title: Mutational and Metal Binding Analysis of the Endonuclease Domain of the Influenza Virus Polymerase PA Subunit ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00995-10

    Isothermal titration calorimetry of wild-type PA-Nter with metal ions. (A) MnCl 2 at 0.3 mM was added to 60 μM PA-Nter at 25°C in 20 mM Tris-HCl (pH 8.0) plus 100 mM NaCl; (B) MgCl 2 at 9 mM was added to 60 μM PA-Nter at 25°C
    Figure Legend Snippet: Isothermal titration calorimetry of wild-type PA-Nter with metal ions. (A) MnCl 2 at 0.3 mM was added to 60 μM PA-Nter at 25°C in 20 mM Tris-HCl (pH 8.0) plus 100 mM NaCl; (B) MgCl 2 at 9 mM was added to 60 μM PA-Nter at 25°C

    Techniques Used: Isothermal Titration Calorimetry

    16) Product Images from "Interaction between the H2 Sensor HupUV and the Histidine Kinase HupT Controls HupSL Hydrogenase Synthesis in Rhodobacter capsulatus"

    Article Title: Interaction between the H2 Sensor HupUV and the Histidine Kinase HupT Controls HupSL Hydrogenase Synthesis in Rhodobacter capsulatus

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.24.7111-7119.2003

    In vitro interaction between the HupUV and HupT proteins. The proteins (40 pmol of HupUV and 120 pmol of HupT) were incubated for 15 min at 30°C in a buffer containing 20 mM Tris-HCl (pH 8), 0.15 M NaCl, and 10% glycerol. The proteins were then run on a native 7.5% acrylamide gel in 0.5× Laemmli buffer, and the gel was revealed either by hydrogenase activity staining (A) or with anti-His 6 tag antibodies (B). (C) Effect of H 2 on the HupUV-HupT interaction. HupUV was first incubated with H 2 during 15 min, and then HupT was added, and the incubation lasted for 15 additional min before the complexes were loaded on the gel.
    Figure Legend Snippet: In vitro interaction between the HupUV and HupT proteins. The proteins (40 pmol of HupUV and 120 pmol of HupT) were incubated for 15 min at 30°C in a buffer containing 20 mM Tris-HCl (pH 8), 0.15 M NaCl, and 10% glycerol. The proteins were then run on a native 7.5% acrylamide gel in 0.5× Laemmli buffer, and the gel was revealed either by hydrogenase activity staining (A) or with anti-His 6 tag antibodies (B). (C) Effect of H 2 on the HupUV-HupT interaction. HupUV was first incubated with H 2 during 15 min, and then HupT was added, and the incubation lasted for 15 additional min before the complexes were loaded on the gel.

    Techniques Used: In Vitro, Incubation, Acrylamide Gel Assay, Activity Assay, Staining

    17) Product Images from "ITC Analysis of Ligand Binding to PreQ1 Riboswitches"

    Article Title: ITC Analysis of Ligand Binding to PreQ1 Riboswitches

    Journal: Methods in enzymology

    doi: 10.1016/B978-0-12-801122-5.00018-0

    Representative isotherm and binding-model fit for a preQ 1 -I type III riboswitch. ITC was performed at 25 °C in 0.0060 M MgCl 2 , 0.10 M NaCl, and 0.050 M Na-HEPES pH 7.0. The wild type riboswitch was in the cell at 8.74 μM; preQ 1 was in the syringe at a concentration 10-fold higher than the RNA. The c value is 112. The parameters obtained from a “One Set of Sites” binding model are shown as text in the inset DeltaH window. The values are: the binding stoichiometry, N = 0.93; K A = 1.39 x 10 7 M −1 ; Δ H = −25.39 x 10 3 cal/mol; and ΔS = −52.4 cal/(mol K).
    Figure Legend Snippet: Representative isotherm and binding-model fit for a preQ 1 -I type III riboswitch. ITC was performed at 25 °C in 0.0060 M MgCl 2 , 0.10 M NaCl, and 0.050 M Na-HEPES pH 7.0. The wild type riboswitch was in the cell at 8.74 μM; preQ 1 was in the syringe at a concentration 10-fold higher than the RNA. The c value is 112. The parameters obtained from a “One Set of Sites” binding model are shown as text in the inset DeltaH window. The values are: the binding stoichiometry, N = 0.93; K A = 1.39 x 10 7 M −1 ; Δ H = −25.39 x 10 3 cal/mol; and ΔS = −52.4 cal/(mol K).

    Techniques Used: Binding Assay, Concentration Assay

    18) Product Images from "Determination of leukotriene A4 stabilization by S100A8/A9 proteins using mass spectrometry"

    Article Title: Determination of leukotriene A4 stabilization by S100A8/A9 proteins using mass spectrometry

    Journal:

    doi: 10.1194/jlr.M900017-JLR200

    A: Western blot for S100A8 and S100A9 proteins in the isolated FPLC fractions of human PMN cytosol. FPLC fractions collected using 0.1 M PBS w/ 0.2 M NaCl at 1 ml/min. Proteins separated using 4 → 20% Tris-HCl gels. B: Coomassie gel staining of
    Figure Legend Snippet: A: Western blot for S100A8 and S100A9 proteins in the isolated FPLC fractions of human PMN cytosol. FPLC fractions collected using 0.1 M PBS w/ 0.2 M NaCl at 1 ml/min. Proteins separated using 4 → 20% Tris-HCl gels. B: Coomassie gel staining of

    Techniques Used: Western Blot, Isolation, Fast Protein Liquid Chromatography, Staining

    19) Product Images from "Molecular characterization of pyridoxine 5′-phosphate oxidase and its pathogenic forms associated with neonatal epileptic encephalopathy"

    Article Title: Molecular characterization of pyridoxine 5′-phosphate oxidase and its pathogenic forms associated with neonatal epileptic encephalopathy

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-70598-7

    Physical properties of PNPO variant forms. Only results from the wild type (black lines), G118R (red lines) and X262Q (blue lines) PNPO enzymes are shown. All the other variants have the same characteristics as the wild type enzyme and therefore are not reported. ( a ) Size exclusion chromatography analyses on PNPO variants. Elution profiles were obtained in 20 mM potassium-phosphate buffer, pH 7.6, containing 150 mM NaCl and 5 mM 2-mercaptoethanol. Far-UV CD ( b ) and absorption ( c ) spectra of wild type, G118R and X262Q PNPO enzymes are shown. Inset in panel C is an expanded view of the visible region of the same absorption spectra, showing absorption bands due to FMN. For each protein sample three independent preparations were analysed, although in the figure a single spectrum is shown of wild type (71% saturation with respect to FMN), G118R (30% saturation) and X262Q (10% saturation). All spectra were measured in 20 mM potassium-phosphate buffer, pH 7.6. Images were generated using the software Prism 8 (GraphPad; https://www.graphpad.com/scientific-software/prism/ ).
    Figure Legend Snippet: Physical properties of PNPO variant forms. Only results from the wild type (black lines), G118R (red lines) and X262Q (blue lines) PNPO enzymes are shown. All the other variants have the same characteristics as the wild type enzyme and therefore are not reported. ( a ) Size exclusion chromatography analyses on PNPO variants. Elution profiles were obtained in 20 mM potassium-phosphate buffer, pH 7.6, containing 150 mM NaCl and 5 mM 2-mercaptoethanol. Far-UV CD ( b ) and absorption ( c ) spectra of wild type, G118R and X262Q PNPO enzymes are shown. Inset in panel C is an expanded view of the visible region of the same absorption spectra, showing absorption bands due to FMN. For each protein sample three independent preparations were analysed, although in the figure a single spectrum is shown of wild type (71% saturation with respect to FMN), G118R (30% saturation) and X262Q (10% saturation). All spectra were measured in 20 mM potassium-phosphate buffer, pH 7.6. Images were generated using the software Prism 8 (GraphPad; https://www.graphpad.com/scientific-software/prism/ ).

    Techniques Used: Variant Assay, Size-exclusion Chromatography, Generated, Software

    20) Product Images from "Comparison of Biochemical Properties of HIV-1 and HIV-2 Capsid Proteins"

    Article Title: Comparison of Biochemical Properties of HIV-1 and HIV-2 Capsid Proteins

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.01082

    Comparative analysis of several CA proteins for their in vitro polymerization properties. CA-polymerization was performed in vitro and monitored by OD at 350 nm as described in MATERIALS AND METHODS. (A) Polymerization of NL4-3 CA (NLCA), GL-AN CA (GLCA), and NL/GL CA (NL/GL) proteins for 4 h at various NaCl concentrations. The chimeric clone NL/GL has the sequence encoding the NTD of NL4-3 CA and the linker domain/CTD of GL-AN CA ( Figure 1A ). (B) Polymerization kinetics of NL and NL/GL CA proteins (1.5 M NaCl). (C) Polymerization of NL and GL32NL CA proteins for 4 h at various NaCl concentrations. GL32NL is a chimeric NLCA-derivative clone which has the sequence encoding the very N-terminal region of GL-AN CA (Pro1-Phe32 in Figure 1A ).
    Figure Legend Snippet: Comparative analysis of several CA proteins for their in vitro polymerization properties. CA-polymerization was performed in vitro and monitored by OD at 350 nm as described in MATERIALS AND METHODS. (A) Polymerization of NL4-3 CA (NLCA), GL-AN CA (GLCA), and NL/GL CA (NL/GL) proteins for 4 h at various NaCl concentrations. The chimeric clone NL/GL has the sequence encoding the NTD of NL4-3 CA and the linker domain/CTD of GL-AN CA ( Figure 1A ). (B) Polymerization kinetics of NL and NL/GL CA proteins (1.5 M NaCl). (C) Polymerization of NL and GL32NL CA proteins for 4 h at various NaCl concentrations. GL32NL is a chimeric NLCA-derivative clone which has the sequence encoding the very N-terminal region of GL-AN CA (Pro1-Phe32 in Figure 1A ).

    Techniques Used: In Vitro, Sequencing

    Thermal stability of CA NTD proteins derived from HIV-1 NL4-3, and HIV-2 GL-AN. The thermal stability of NLCA and GLCA NTD proteins in the presence of 250 mM NaCl was determined by DSF as described in the Section “Materials and Methods”. SYPRO orange fluorescence intensity (FI) at varying temperatures (upper panel) and derivative melt curves calculated by differences in FI at each temperature (lower panel) are shown. Peak temperatures in the curves (dFI/dT) were considered as Tm.
    Figure Legend Snippet: Thermal stability of CA NTD proteins derived from HIV-1 NL4-3, and HIV-2 GL-AN. The thermal stability of NLCA and GLCA NTD proteins in the presence of 250 mM NaCl was determined by DSF as described in the Section “Materials and Methods”. SYPRO orange fluorescence intensity (FI) at varying temperatures (upper panel) and derivative melt curves calculated by differences in FI at each temperature (lower panel) are shown. Peak temperatures in the curves (dFI/dT) were considered as Tm.

    Techniques Used: Derivative Assay, Fluorescence

    Thermal stability of CA NTD proteins at various NaCl concentrations. The thermal stability of NLCA and GLCA NTD proteins at various NaCl concentrations was evaluated as described in the legend to Figure 6 . FI values and melt curves ( A,B , respectively), and Tm shifts induced by NaCl (C) are shown.
    Figure Legend Snippet: Thermal stability of CA NTD proteins at various NaCl concentrations. The thermal stability of NLCA and GLCA NTD proteins at various NaCl concentrations was evaluated as described in the legend to Figure 6 . FI values and melt curves ( A,B , respectively), and Tm shifts induced by NaCl (C) are shown.

    Techniques Used:

    21) Product Images from "D’ domain region Arg782-Cys799 of von Willebrand factor contributes to factor VIII binding"

    Article Title: D’ domain region Arg782-Cys799 of von Willebrand factor contributes to factor VIII binding

    Journal: Haematologica

    doi: 10.3324/haematol.2019.221994

    The FVIII binding efficiency of D’-D3 Leu786Ala. (A) Part of the crystal structure of D’-D3 (PDB entry: 6n29)30 with a zoom-in of the helical region comprising the residues 786-Leu-Glu-Cys-789. (B) Multiple concentrations of D’-D3 Leu786Ala were passed over coagulation factor VIII (FVIII) that was immobilized via antibody EL14 to the surface of a CM5 sensor chip. The binding response is indicated as response units (RU) and was assessed in 20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM CaCl 2 , 0.05% (v/v) Tween 20 at a flow rate of 30 μL/min at 25°C. (C) FVIII was pre-incubated with increasing concentrations of D’-D3 and D’-D3 Leu786Ala in a buffer comprising 50 mM Tris (pH 7.4), 150 mM NaCl, 5mM CaCl 2 , 2% human serum albumin and 0.1% Tween 20 at 37°C. The protein mixtures were next incubated with immobilized von Willebrand factor (VWF) in the same buffer. Residual FVIII binding to immobilized VWF was assessed employing HRP-conjugated CAg12 antibody as described in the methods. Data represents mean ± standard deviation (SD) of three independent experiments.
    Figure Legend Snippet: The FVIII binding efficiency of D’-D3 Leu786Ala. (A) Part of the crystal structure of D’-D3 (PDB entry: 6n29)30 with a zoom-in of the helical region comprising the residues 786-Leu-Glu-Cys-789. (B) Multiple concentrations of D’-D3 Leu786Ala were passed over coagulation factor VIII (FVIII) that was immobilized via antibody EL14 to the surface of a CM5 sensor chip. The binding response is indicated as response units (RU) and was assessed in 20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM CaCl 2 , 0.05% (v/v) Tween 20 at a flow rate of 30 μL/min at 25°C. (C) FVIII was pre-incubated with increasing concentrations of D’-D3 and D’-D3 Leu786Ala in a buffer comprising 50 mM Tris (pH 7.4), 150 mM NaCl, 5mM CaCl 2 , 2% human serum albumin and 0.1% Tween 20 at 37°C. The protein mixtures were next incubated with immobilized von Willebrand factor (VWF) in the same buffer. Residual FVIII binding to immobilized VWF was assessed employing HRP-conjugated CAg12 antibody as described in the methods. Data represents mean ± standard deviation (SD) of three independent experiments.

    Techniques Used: Binding Assay, Coagulation, Chromatin Immunoprecipitation, Incubation, Standard Deviation

    D’-D3 variants in competition with immobilized von Willebrand factor for binding FVIII. Coagulation factor VIII (FVIII) was incubated with increasing concentrations of the indicated D’-D3 variants in a buffer comprising 50 mM Tris (pH 7.4), 150 mM NaCl, 5mM CaCl 2 , 2% human serum albumin and 0.1% Tween 20 at 37°C. The protein mixtures were next incubated with immobilized von Willebrand factor (VWF) in the same buffer. Residual FVIII binding to immobilized VWF was assessed employing HRP-conjugated CAg12 antibody as described in the methods. Data represents mean ± standard deviation (SD) of three independent experiments.
    Figure Legend Snippet: D’-D3 variants in competition with immobilized von Willebrand factor for binding FVIII. Coagulation factor VIII (FVIII) was incubated with increasing concentrations of the indicated D’-D3 variants in a buffer comprising 50 mM Tris (pH 7.4), 150 mM NaCl, 5mM CaCl 2 , 2% human serum albumin and 0.1% Tween 20 at 37°C. The protein mixtures were next incubated with immobilized von Willebrand factor (VWF) in the same buffer. Residual FVIII binding to immobilized VWF was assessed employing HRP-conjugated CAg12 antibody as described in the methods. Data represents mean ± standard deviation (SD) of three independent experiments.

    Techniques Used: Binding Assay, Coagulation, Incubation, Standard Deviation

    22) Product Images from "The role of the N-terminus in determining metal specific responses in the E. coli Ni- and Co-responsive metalloregulator, RcnR"

    Article Title: The role of the N-terminus in determining metal specific responses in the E. coli Ni- and Co-responsive metalloregulator, RcnR

    Journal: Journal of the American Chemical Society

    doi: 10.1021/ja300834b

    Metal complexes of wild-type RcnR in buffer with 20 mM Hepes, 300 mM NaCl, and 10 % glycerol at pH 7.0. Left: Fourier-filtered XAS data (colored lines) and best fits (black lines) from . Right: Unfiltered k 3 -weighted EXAFS spectra and fits.
    Figure Legend Snippet: Metal complexes of wild-type RcnR in buffer with 20 mM Hepes, 300 mM NaCl, and 10 % glycerol at pH 7.0. Left: Fourier-filtered XAS data (colored lines) and best fits (black lines) from . Right: Unfiltered k 3 -weighted EXAFS spectra and fits.

    Techniques Used:

    23) Product Images from "Impact of meriolins, a new class of cyclin-dependent kinase inhibitors, on malignant glioma proliferation and neo-angiogenesis"

    Article Title: Impact of meriolins, a new class of cyclin-dependent kinase inhibitors, on malignant glioma proliferation and neo-angiogenesis

    Journal: Neuro-Oncology

    doi: 10.1093/neuonc/nou102

    Inhibitory role of meriolin 15 on U87 and human GBM sc1 xenograft development in nude mice. (A and B) U87 cells (3 × 10 6 cells, 100 µL PBS) were implanted subcutaneously into the right flanks of nude mice. (A) When tumors reached a volume of ∼100–150 mm 3 , mice were treated i.p. every 2 days with meriolin 15 (2 mg/kg/2d, n = 4) or (5 mg/kg/2d, n = 5) or every day (5 mg/kg/d, n = 4) or with vehicle (DMSO in NaCl 0.9%, n = 4) during 22 days. Statistical significance was determined by the Freidman test followed by the Dunn' multiple comparison test. * P
    Figure Legend Snippet: Inhibitory role of meriolin 15 on U87 and human GBM sc1 xenograft development in nude mice. (A and B) U87 cells (3 × 10 6 cells, 100 µL PBS) were implanted subcutaneously into the right flanks of nude mice. (A) When tumors reached a volume of ∼100–150 mm 3 , mice were treated i.p. every 2 days with meriolin 15 (2 mg/kg/2d, n = 4) or (5 mg/kg/2d, n = 5) or every day (5 mg/kg/d, n = 4) or with vehicle (DMSO in NaCl 0.9%, n = 4) during 22 days. Statistical significance was determined by the Freidman test followed by the Dunn' multiple comparison test. * P

    Techniques Used: Mouse Assay

    24) Product Images from "RNase H1 directs origin-specific initiation of DNA replication in human mitochondria"

    Article Title: RNase H1 directs origin-specific initiation of DNA replication in human mitochondria

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007781

    Factors affecting R-loop formation in vitro. A. Purified, recombinant proteins used in the present study visualized by Stain Free SDS-PAGE (Bio-Rad). B. In vitro transcription from LSP with POLRMT (20 nM), TFAM (200 nM) and TFB2M (60 nM). R-loops were formed and detected as described in panel C. TEFM (40 nM) was added to the indicated reactions. Products formed are labeled as followed: PT: transcripts prematurely terminated at CSBII; RC: longer transcripts formed by rolling circle transcription; and R-loops: transcripts unaffected by RNase A treatment (lane 6). The RNA was labeled by [ 32 P]UTP incorporation. C. Reaction scheme for R-loop formation. A pUC18 plasmid containing an LSP insert, including the CSB region (pUC-LSP, S1 Table ) was used. When indicated, the template was treated with topoisomerase I to relax supercoils. In vitro transcription was performed in the presence or absence of TEFM followed by the addition of 300 mM NaCl and RNase A to remove free RNA. D. Effects of mtSSB on in vitro transcription and R-loop formation. Templates used were supercoiled pUC-LSP (lanes 1-6) and as a control, linear pUC-HSP (lanes 7-10, see S1 Table for template sequence). mtSSB concentrations are indicated in nM. HSP RO: Run-off product of HSP transcription; PT: transcripts prematurely terminated at CSBII; and R-loops: transcripts unaffected by RNase A treatment. The ratio of R-loops/CSBII pre-terminated transcripts for each mtSSB concentration is indicated (see Materials and methods ). E. R-loop formation was as in 1C, but without RNase A treatment. Increasing RNase H1 concentrations were added (0, 1, 2, 4, 8, 16 and 32 nM in lanes 1-7). PT indicates transcripts prematurely terminated at CSBII.
    Figure Legend Snippet: Factors affecting R-loop formation in vitro. A. Purified, recombinant proteins used in the present study visualized by Stain Free SDS-PAGE (Bio-Rad). B. In vitro transcription from LSP with POLRMT (20 nM), TFAM (200 nM) and TFB2M (60 nM). R-loops were formed and detected as described in panel C. TEFM (40 nM) was added to the indicated reactions. Products formed are labeled as followed: PT: transcripts prematurely terminated at CSBII; RC: longer transcripts formed by rolling circle transcription; and R-loops: transcripts unaffected by RNase A treatment (lane 6). The RNA was labeled by [ 32 P]UTP incorporation. C. Reaction scheme for R-loop formation. A pUC18 plasmid containing an LSP insert, including the CSB region (pUC-LSP, S1 Table ) was used. When indicated, the template was treated with topoisomerase I to relax supercoils. In vitro transcription was performed in the presence or absence of TEFM followed by the addition of 300 mM NaCl and RNase A to remove free RNA. D. Effects of mtSSB on in vitro transcription and R-loop formation. Templates used were supercoiled pUC-LSP (lanes 1-6) and as a control, linear pUC-HSP (lanes 7-10, see S1 Table for template sequence). mtSSB concentrations are indicated in nM. HSP RO: Run-off product of HSP transcription; PT: transcripts prematurely terminated at CSBII; and R-loops: transcripts unaffected by RNase A treatment. The ratio of R-loops/CSBII pre-terminated transcripts for each mtSSB concentration is indicated (see Materials and methods ). E. R-loop formation was as in 1C, but without RNase A treatment. Increasing RNase H1 concentrations were added (0, 1, 2, 4, 8, 16 and 32 nM in lanes 1-7). PT indicates transcripts prematurely terminated at CSBII.

    Techniques Used: In Vitro, Purification, Recombinant, Staining, SDS Page, Labeling, Plasmid Preparation, Sequencing, Concentration Assay

    25) Product Images from "Bordetella Pertussis Adenylate Cyclase Toxin Does Not Possess a Phospholipase A Activity; Serine 606 and Aspartate 1079 Residues Are Not Involved in Target Cell Delivery of the Adenylyl Cyclase Enzyme Domain"

    Article Title: Bordetella Pertussis Adenylate Cyclase Toxin Does Not Possess a Phospholipase A Activity; Serine 606 and Aspartate 1079 Residues Are Not Involved in Target Cell Delivery of the Adenylyl Cyclase Enzyme Domain

    Journal: Toxins

    doi: 10.3390/toxins10060245

    The purified CyaA toxin is devoid of any detectable phospholipase A1 activity. ( a ) The stock solution of phospholipase A1 from Thermomyces lanuginosus was diluted 1:100 or 1:10,000 in 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 10 mM CaCl 2 (or 20 mM EDTA), and incubated with 500 nM PED-A1, a selective PLA-1 fluorogenic substrate, before the fluorescence emission at 530 nm (excitation at 488 nm) was recorded under a continuous kinetic readout in the microtiter plate. Fluorescence intensities of the cleaved fluorogenic substrate were subtracted from the background and averaged. The values represent the average ± standard deviations derived from three independent experiments performed in triplicate ( n = 9). ( b ) Individual purified CyaA fractions were diluted in buffer to provide a final AC enzyme activity of 1 U/mL. Incubation with 500 nM PED-A1 occurred before fluorescence emission at 530 nm was recorded at indicated time points of t = 1, 5, 10, 15, and 20 min. The values represent the average ± standard deviations derived from three independent experiments performed in triplicate with two independent CyaA preparations ( n = 9). ( c ) The 500 nM PED-A1 substrate was mixed with highly purified CyaA (DEAE + CaM + Phenyl, 1 U/mL) and 1:100 diluted phospholipase A1 from Thermomyces lanuginosus was added. Fluorescence emission at 530 nm was recorded.
    Figure Legend Snippet: The purified CyaA toxin is devoid of any detectable phospholipase A1 activity. ( a ) The stock solution of phospholipase A1 from Thermomyces lanuginosus was diluted 1:100 or 1:10,000 in 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 10 mM CaCl 2 (or 20 mM EDTA), and incubated with 500 nM PED-A1, a selective PLA-1 fluorogenic substrate, before the fluorescence emission at 530 nm (excitation at 488 nm) was recorded under a continuous kinetic readout in the microtiter plate. Fluorescence intensities of the cleaved fluorogenic substrate were subtracted from the background and averaged. The values represent the average ± standard deviations derived from three independent experiments performed in triplicate ( n = 9). ( b ) Individual purified CyaA fractions were diluted in buffer to provide a final AC enzyme activity of 1 U/mL. Incubation with 500 nM PED-A1 occurred before fluorescence emission at 530 nm was recorded at indicated time points of t = 1, 5, 10, 15, and 20 min. The values represent the average ± standard deviations derived from three independent experiments performed in triplicate with two independent CyaA preparations ( n = 9). ( c ) The 500 nM PED-A1 substrate was mixed with highly purified CyaA (DEAE + CaM + Phenyl, 1 U/mL) and 1:100 diluted phospholipase A1 from Thermomyces lanuginosus was added. Fluorescence emission at 530 nm was recorded.

    Techniques Used: Purification, Activity Assay, Incubation, Proximity Ligation Assay, Fluorescence, Derivative Assay, Chick Chorioallantoic Membrane Assay

    CyaA-S606A and CyaA-D1079A toxins exhibit an almost identical specific cytotoxic activity against sheep erythrocytes and mouse macrophages J774A.1 as intact CyaA. Proteins were expressed in E. coli BL-21 cells and purified from urea extracts on DEAE Sepharose. ( a ) Sheep erythrocytes (5 × 10 8 cells/mL) were incubated at 37 °C in the presence of 2 mM calcium (+Ca 2+ ) or 5 mM EDTA (+EDTA) with 1 µg/mL (~0.4 U/mL) of the purified CyaA proteins and after 30 min. Aliquots were used for determination of the cell-associated AC activity (binding) and of the AC activity internalized into erythrocytes and protected against digestion by externally added trypsin (invasive AC). Activities are expressed as percentages of intact CyaA activity and represent average values ± standard deviations from at least three independent determinations performed in duplicate with two different toxin preparations ( n = 6–8). ( b ) Sheep erythrocytes (5 × 10 8 cells/mL) in Tris 50 mM, NaCl 150 mM, CaCl 2 2 mM, pH 7.4 buffer (TNC) were incubated at 37 °C with intact CyaA or with its mutant variants (10 µg/mL). Hemolytic activity was measured as the amount of released hemoglobin by photometric determination (A 541 ), ( n = 3). ( c ) Binding of intact CyaA or its mutant variants to J774A.1 cells (1 × 10 6 ) was determined as the amount of total cell-associated AC enzyme activity upon incubation of cells with 1 µg/mL (~0.4 U/mL) of the protein for 30 min at 4 °C. cAMP intoxication was assessed by determining the intracellular concentration of cAMP generated in cells after 30 min of incubation of J774A.1 cells (2 × 10 5 ) with four different toxin concentrations from within the linear range of the dose-response curve (100, 50, 25, and 12.5 ng/mL, i.e., ~40, 20, 10, 5 mU/mL, respectively). Activities are expressed as percentages of intact CyaA activity and represent average values ± standard deviations from at least three independent determinations performed in duplicate with two different toxin preparations ( n = 6–8).
    Figure Legend Snippet: CyaA-S606A and CyaA-D1079A toxins exhibit an almost identical specific cytotoxic activity against sheep erythrocytes and mouse macrophages J774A.1 as intact CyaA. Proteins were expressed in E. coli BL-21 cells and purified from urea extracts on DEAE Sepharose. ( a ) Sheep erythrocytes (5 × 10 8 cells/mL) were incubated at 37 °C in the presence of 2 mM calcium (+Ca 2+ ) or 5 mM EDTA (+EDTA) with 1 µg/mL (~0.4 U/mL) of the purified CyaA proteins and after 30 min. Aliquots were used for determination of the cell-associated AC activity (binding) and of the AC activity internalized into erythrocytes and protected against digestion by externally added trypsin (invasive AC). Activities are expressed as percentages of intact CyaA activity and represent average values ± standard deviations from at least three independent determinations performed in duplicate with two different toxin preparations ( n = 6–8). ( b ) Sheep erythrocytes (5 × 10 8 cells/mL) in Tris 50 mM, NaCl 150 mM, CaCl 2 2 mM, pH 7.4 buffer (TNC) were incubated at 37 °C with intact CyaA or with its mutant variants (10 µg/mL). Hemolytic activity was measured as the amount of released hemoglobin by photometric determination (A 541 ), ( n = 3). ( c ) Binding of intact CyaA or its mutant variants to J774A.1 cells (1 × 10 6 ) was determined as the amount of total cell-associated AC enzyme activity upon incubation of cells with 1 µg/mL (~0.4 U/mL) of the protein for 30 min at 4 °C. cAMP intoxication was assessed by determining the intracellular concentration of cAMP generated in cells after 30 min of incubation of J774A.1 cells (2 × 10 5 ) with four different toxin concentrations from within the linear range of the dose-response curve (100, 50, 25, and 12.5 ng/mL, i.e., ~40, 20, 10, 5 mU/mL, respectively). Activities are expressed as percentages of intact CyaA activity and represent average values ± standard deviations from at least three independent determinations performed in duplicate with two different toxin preparations ( n = 6–8).

    Techniques Used: Activity Assay, Purification, Incubation, Binding Assay, Mutagenesis, Concentration Assay, Generated

    26) Product Images from "Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: Role in osteoclast-mediated NK cell activation"

    Article Title: Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: Role in osteoclast-mediated NK cell activation

    Journal: Oncotarget

    doi:

    In vivo injection of ZOL triggered significant IFN-γ and IL-6 secretion by bone marrow-derived cells but inhibited secretion by gingival cells A. Female Balb/c mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction, the maxillary palatal mucosa of 0.9% NACL solution-injected control mice showed complete wound healing, whereas tissue swelling (dotted line) around the tooth extraction site remained in ZOL-injected mice. The maxillary bone of FAM-ZOL-injected mice showed the strong fluorescence signal, while the remaining molars (M1, M2, M3) were absent of fluorescence. The visibly reduced fluorescence was observed in the tooth extraction sockets (*) and the adjacent palatal bone area (dotted line and arrowheads) B. Hematoxylin and eosin (H E) stained histological sections of ZOL-injected mice showed significant inflammatory cell infiltration (Inflam) in the palatal/gingival soft tissue one week after tooth extraction. There were signs of abnormal epithelial hyperplasia (Epi.Hyp.: arrows). The extraction socket (*) exhibited new bone formation. TRAP staining revealed a number of osteoclasts (vertical arrows) on the surface of palatal bone (Palat.Bone) as well as in the extraction socket (*). TRAP + multinuclear cells were found within the inflammation area (arrowheads). In non-decalcified cross-section of FAM-ZOL-injected mice, FAM-positive large cells (arrows) were found on the palatal bone as well as away from the bone surface (arrowheads). Taken together, these cells in the inflammatory area were thought to be ZOL-affected osteoclasts C. Immunohistology for cytokeratin 14 (CK14) was performed as described in the Materials and Method section, and confirmed oral epithelial hyperplasia in the palatal/gingival tissue of ZOL-injected mice (Epi.Hyp.: arrows). Epithelial hyperplasia was primarily observed within the strong inflammatory area D. Female B6 mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction the surface expression of CD45 − FITC + CD3 − PE or CD45 − FITC + DX5 − PE on cells obtained from gingival tissues. E. and bone marrow F. were assessed with flow cytometric analysis after staining with the respective PE- and FITC-conjugated antibodies. Isotype cont rol antibodies were used as control. Right upper quadrant represent the percentage of CD3 and DX5 positive cells within the CD45 population. B6 mice underwent experimental treatment as described in Fig. 7D . Gingival tissues obtained from ZOL and NACL injected animals were digested as described in Materials and Method. Dissociated cells obtained from gingival tissues were cultured in the presence of IL-2 (10,000 units/ml). Supernatants were harvested 5 days after IL-2 treatment. The mean for ( n = 4) is shown for each set in the figure. The difference between ZOL and NACL injected gingivae is significant at a P
    Figure Legend Snippet: In vivo injection of ZOL triggered significant IFN-γ and IL-6 secretion by bone marrow-derived cells but inhibited secretion by gingival cells A. Female Balb/c mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction, the maxillary palatal mucosa of 0.9% NACL solution-injected control mice showed complete wound healing, whereas tissue swelling (dotted line) around the tooth extraction site remained in ZOL-injected mice. The maxillary bone of FAM-ZOL-injected mice showed the strong fluorescence signal, while the remaining molars (M1, M2, M3) were absent of fluorescence. The visibly reduced fluorescence was observed in the tooth extraction sockets (*) and the adjacent palatal bone area (dotted line and arrowheads) B. Hematoxylin and eosin (H E) stained histological sections of ZOL-injected mice showed significant inflammatory cell infiltration (Inflam) in the palatal/gingival soft tissue one week after tooth extraction. There were signs of abnormal epithelial hyperplasia (Epi.Hyp.: arrows). The extraction socket (*) exhibited new bone formation. TRAP staining revealed a number of osteoclasts (vertical arrows) on the surface of palatal bone (Palat.Bone) as well as in the extraction socket (*). TRAP + multinuclear cells were found within the inflammation area (arrowheads). In non-decalcified cross-section of FAM-ZOL-injected mice, FAM-positive large cells (arrows) were found on the palatal bone as well as away from the bone surface (arrowheads). Taken together, these cells in the inflammatory area were thought to be ZOL-affected osteoclasts C. Immunohistology for cytokeratin 14 (CK14) was performed as described in the Materials and Method section, and confirmed oral epithelial hyperplasia in the palatal/gingival tissue of ZOL-injected mice (Epi.Hyp.: arrows). Epithelial hyperplasia was primarily observed within the strong inflammatory area D. Female B6 mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction the surface expression of CD45 − FITC + CD3 − PE or CD45 − FITC + DX5 − PE on cells obtained from gingival tissues. E. and bone marrow F. were assessed with flow cytometric analysis after staining with the respective PE- and FITC-conjugated antibodies. Isotype cont rol antibodies were used as control. Right upper quadrant represent the percentage of CD3 and DX5 positive cells within the CD45 population. B6 mice underwent experimental treatment as described in Fig. 7D . Gingival tissues obtained from ZOL and NACL injected animals were digested as described in Materials and Method. Dissociated cells obtained from gingival tissues were cultured in the presence of IL-2 (10,000 units/ml). Supernatants were harvested 5 days after IL-2 treatment. The mean for ( n = 4) is shown for each set in the figure. The difference between ZOL and NACL injected gingivae is significant at a P

    Techniques Used: In Vivo, Injection, Derivative Assay, Mouse Assay, IV Injection, Fluorescence, Staining, Expressing, Flow Cytometry, Cell Culture

    27) Product Images from "Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation"

    Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

    Journal: Molecular Genetics & Genomic Medicine

    doi: 10.1002/mgg3.3

    Ligand binding can affect the thermal stability of PMM2. Heat-induced melting profile of wild-type PMM2 (A and C) and F119L-PMM2 (B and D) were recorded by thermal shift assay and by circular dichroism. For thermal shift assay, the proteins (0.2 mg/mL) were equilibrated in buffer (HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5) containing Sypro Orange2.5X and the appropriate ligands: MgCl 2 5 mmol/L, EDTA 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L + vanadate 0.5 mmol/L, Glu-1,6-P 0.5 mmol/L + MgCl 2 5 mmol/L. The samples were distributed in 96-well PCR plates, the plates were sealed, and heated from 25 to 80° at 0.5°C/min. The experiment was run on an iCycler iQ Real Time PCR Detection System. An excitation wavelength of 490 nm and an emission wavelength of 575 nm were used to collect the data. When the melting profile was obtained by circular dichroism the proteins (0.2 mg/mL) were equilibrated in the same buffer in the presence of MgCl 2 1 mmol/L or EDTA 5 mmol/L. The signal at 222 nm was recorded while temperature was increased at 0.5°C/min from 20°C. The raw data were corrected by taking into account the slopes of the pre- and post-transition baselines, then they were normalized.
    Figure Legend Snippet: Ligand binding can affect the thermal stability of PMM2. Heat-induced melting profile of wild-type PMM2 (A and C) and F119L-PMM2 (B and D) were recorded by thermal shift assay and by circular dichroism. For thermal shift assay, the proteins (0.2 mg/mL) were equilibrated in buffer (HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5) containing Sypro Orange2.5X and the appropriate ligands: MgCl 2 5 mmol/L, EDTA 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L + vanadate 0.5 mmol/L, Glu-1,6-P 0.5 mmol/L + MgCl 2 5 mmol/L. The samples were distributed in 96-well PCR plates, the plates were sealed, and heated from 25 to 80° at 0.5°C/min. The experiment was run on an iCycler iQ Real Time PCR Detection System. An excitation wavelength of 490 nm and an emission wavelength of 575 nm were used to collect the data. When the melting profile was obtained by circular dichroism the proteins (0.2 mg/mL) were equilibrated in the same buffer in the presence of MgCl 2 1 mmol/L or EDTA 5 mmol/L. The signal at 222 nm was recorded while temperature was increased at 0.5°C/min from 20°C. The raw data were corrected by taking into account the slopes of the pre- and post-transition baselines, then they were normalized.

    Techniques Used: Ligand Binding Assay, Thermal Shift Assay, Gas Chromatography, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Specific activity of PMM2 depends on glucose-1,6-bisphosphate concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glu-1-P (0.04 or 0.60 mmol/L), and yeast glucose 6-phosphate dehydrogenase 10 μg/mL, while Glc-1,6-P 2 was changed in the range 0–80 μmol/L. Enzymes concentrations were 107 nmol/L for wt-PMM2 and 73 nmol/L for F119L-PMM2. The hyperbolic dependence of velocity on the activator concentration was fitted using Michaelis and Menten equation to evaluate EC50.
    Figure Legend Snippet: Specific activity of PMM2 depends on glucose-1,6-bisphosphate concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glu-1-P (0.04 or 0.60 mmol/L), and yeast glucose 6-phosphate dehydrogenase 10 μg/mL, while Glc-1,6-P 2 was changed in the range 0–80 μmol/L. Enzymes concentrations were 107 nmol/L for wt-PMM2 and 73 nmol/L for F119L-PMM2. The hyperbolic dependence of velocity on the activator concentration was fitted using Michaelis and Menten equation to evaluate EC50.

    Techniques Used: Activity Assay, Concentration Assay, Gas Chromatography

    Long-term stability of F119L-PMM2. F119L-PMM2 (0.027 mmol/L of monomer equivalents) was equilibrated in HEPES 50 mmol/L pH 7.1 containing NaCl 150 mmol/L. Aliquots containing 1.6 μg of protein were taken at known incubation time and diluted immediately to assay the residual activity with Glc-1-P under standard conditions. (A) Results obtained at 37°C in the presence of EDTA 0.1 mmol/L or MgCl 2 5 mmol/L. (B) Results obtained at 44°C in the presence of MgCl 2 5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L and vanadate 0.5 mmol/L or MgCl 2 5 mmol/L plus Glu-1,6-P 2 0.5 mmol/L
    Figure Legend Snippet: Long-term stability of F119L-PMM2. F119L-PMM2 (0.027 mmol/L of monomer equivalents) was equilibrated in HEPES 50 mmol/L pH 7.1 containing NaCl 150 mmol/L. Aliquots containing 1.6 μg of protein were taken at known incubation time and diluted immediately to assay the residual activity with Glc-1-P under standard conditions. (A) Results obtained at 37°C in the presence of EDTA 0.1 mmol/L or MgCl 2 5 mmol/L. (B) Results obtained at 44°C in the presence of MgCl 2 5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L and vanadate 0.5 mmol/L or MgCl 2 5 mmol/L plus Glu-1,6-P 2 0.5 mmol/L

    Techniques Used: Incubation, Activity Assay, Gas Chromatography

    Ligand binding can increase the resistance to proteases of PMM2. Purified wild-type PMM2 and F119L-PMM2 (A) were incubated (0.5 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w) for 1 or 2 h at 37°C before they were analyzed (5 μg of each sample) by SDS-PAGE. Purified F119L-PMM2 (B) was incubated (0.2 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w), in the presence of no ligands, Glc-1,6-P 2 0.5 mmol/L or Glu-6-P 0.5 mmol/L plus vanadate 0.5 mmol/L, for 2 h at 37°C before they were analyzed (2 μg of each sample) by SDS-PAGE. The protein bands were visualized by Coomassie blue staining and the intensity of the bands quantified. The not-digested protein was quantified and expressed as percentage of the starting material (no protease panel A; time 0 panel B).
    Figure Legend Snippet: Ligand binding can increase the resistance to proteases of PMM2. Purified wild-type PMM2 and F119L-PMM2 (A) were incubated (0.5 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w) for 1 or 2 h at 37°C before they were analyzed (5 μg of each sample) by SDS-PAGE. Purified F119L-PMM2 (B) was incubated (0.2 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w), in the presence of no ligands, Glc-1,6-P 2 0.5 mmol/L or Glu-6-P 0.5 mmol/L plus vanadate 0.5 mmol/L, for 2 h at 37°C before they were analyzed (2 μg of each sample) by SDS-PAGE. The protein bands were visualized by Coomassie blue staining and the intensity of the bands quantified. The not-digested protein was quantified and expressed as percentage of the starting material (no protease panel A; time 0 panel B).

    Techniques Used: Ligand Binding Assay, Purification, Incubation, SDS Page, Gas Chromatography, Staining

    Ligand binding affects the quaternary structure of PMM2. Wild-type PMM2 (0.010 mg) and F119L-PMM2 (0.0065 mg) were subjected to size exclusion chromatography on BioSep-SEC-S3000 column equilibrated in HEPES 20 mmol/L pH 7.5, NaCl 150 mmol/L, MgCl 2 5 mmol/L (long dashed line for the wild-type PMM2 or short dashed line for F119L-PMM2) or in the same buffer containing Glc-6-P 0.5 mmol/L and vanadate 0.1 mmol/L (continuous line for wild-type PMM2 or dotted line for F119L-PMM2). The chromatography was run at room temperature at 0.5 mL/min.
    Figure Legend Snippet: Ligand binding affects the quaternary structure of PMM2. Wild-type PMM2 (0.010 mg) and F119L-PMM2 (0.0065 mg) were subjected to size exclusion chromatography on BioSep-SEC-S3000 column equilibrated in HEPES 20 mmol/L pH 7.5, NaCl 150 mmol/L, MgCl 2 5 mmol/L (long dashed line for the wild-type PMM2 or short dashed line for F119L-PMM2) or in the same buffer containing Glc-6-P 0.5 mmol/L and vanadate 0.1 mmol/L (continuous line for wild-type PMM2 or dotted line for F119L-PMM2). The chromatography was run at room temperature at 0.5 mL/min.

    Techniques Used: Ligand Binding Assay, Size-exclusion Chromatography, Gas Chromatography, Chromatography

    Specific activity of F119L-PMM2 depends on enzyme concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L, and yeast glucose 6-phosphate dehydrogenase 10 μg/mL. The reaction mixture also contained BSA at 0.5 mg/mL. Three sets of experiments were carried out in the presence of 0.04, 0.16, or 0.6 mmol/L Glc-1-P and the F119L-PMM2 concentration changed in the range 10–240 nmol/L (monomer equivalents).
    Figure Legend Snippet: Specific activity of F119L-PMM2 depends on enzyme concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L, and yeast glucose 6-phosphate dehydrogenase 10 μg/mL. The reaction mixture also contained BSA at 0.5 mg/mL. Three sets of experiments were carried out in the presence of 0.04, 0.16, or 0.6 mmol/L Glc-1-P and the F119L-PMM2 concentration changed in the range 10–240 nmol/L (monomer equivalents).

    Techniques Used: Activity Assay, Concentration Assay, Gas Chromatography

    Specific activity of wt-PMM2 changes as a function of protein concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L and yeast glucose 6-phosphate dehydrogenase 0.010 mg/mL. The reaction mixture also contained Glc-1-P 0.020 mmol/L and BSA 0.5 mg/mL. The wt-PMM2 concentration changed in the range 2–110 nmol/L (monomer equivalents).
    Figure Legend Snippet: Specific activity of wt-PMM2 changes as a function of protein concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L and yeast glucose 6-phosphate dehydrogenase 0.010 mg/mL. The reaction mixture also contained Glc-1-P 0.020 mmol/L and BSA 0.5 mg/mL. The wt-PMM2 concentration changed in the range 2–110 nmol/L (monomer equivalents).

    Techniques Used: Activity Assay, Protein Concentration, Gas Chromatography, Concentration Assay

    28) Product Images from "HOS15 Interacts with the Histone Deacetylase HDA9 and the Evening Complex to Epigenetically Regulate the Floral Activator GIGANTEA [OPEN]"

    Article Title: HOS15 Interacts with the Histone Deacetylase HDA9 and the Evening Complex to Epigenetically Regulate the Floral Activator GIGANTEA [OPEN]

    Journal: The Plant Cell

    doi: 10.1105/tpc.18.00721

    HOS15 Associates with EC Components. (A) BiFC analysis of proteins transiently expressed in N. benthamiana leaves. VN and VC indicate the N and C termini, respectively, of Venus (eYFP). Fluorescence (left panels) was detected by confocal microscopy. Right panels are overlay of fluorescence and differential interference contest images. Bars = 100 μm. (B) HOS15 interacts with ELF3. Wild type (10 d old) and hos15-2 plants were cross-linked in 1% formaldehyde to preserve in vivo interaction after harvesting plant material. Total protein extracts were immunoprecipitated with α-HOS15 antibody and resolved by SDS-PAGE. Immunoblots were probed with α-HOS15, or with α-ELF3 to detect ELF3. *Nonspecific bands. (C) HOS15 forms a complex with EC components. N. benthamiana plants were infiltrated with Agrobacterium harboring 35S:HA-LUX, 35S:FLAG-HOS15, and 35S:MYC-ELF3 for transient expression. The proteins were immunoprecipitated with α-HA antibody and resolved by SDS-PAGE. The immunoblots were probed with α-HA to detect LUX, α-HOS15, or α-MYC to detect ELF3. (D) Gel-filtration analysis of HOS15, LUX, and ELF3. Total proteins extracted from 2-week-old wild type plants were fractionated by size-exclusion chromatography using a Superdex 200 10/300GL column equilibrated with elution buffer [50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 0.02% sodium azide]. The samples were separated by SDS-PAGE and subjected to immunoblotting analysis using α-HOS15, α-LUX, or α-ELF3. Aliquots of total protein extracts from wild type, input after ammonium sulfate precipitation of wild type total extracts, and total protein extracts individually isolated from elf3-1 , hos15-2 , or pcl1-1 mutants were used as input controls. Molecular mass markers (ferritin, 440 kD; β-amylase, 200 kD; alcohol dehydrogenase, 150 kD; BSA, 67 kD) were independently eluted using the same equilibrated column. Total, total protein extract; Input, input after ammonium sulfate precipitation.
    Figure Legend Snippet: HOS15 Associates with EC Components. (A) BiFC analysis of proteins transiently expressed in N. benthamiana leaves. VN and VC indicate the N and C termini, respectively, of Venus (eYFP). Fluorescence (left panels) was detected by confocal microscopy. Right panels are overlay of fluorescence and differential interference contest images. Bars = 100 μm. (B) HOS15 interacts with ELF3. Wild type (10 d old) and hos15-2 plants were cross-linked in 1% formaldehyde to preserve in vivo interaction after harvesting plant material. Total protein extracts were immunoprecipitated with α-HOS15 antibody and resolved by SDS-PAGE. Immunoblots were probed with α-HOS15, or with α-ELF3 to detect ELF3. *Nonspecific bands. (C) HOS15 forms a complex with EC components. N. benthamiana plants were infiltrated with Agrobacterium harboring 35S:HA-LUX, 35S:FLAG-HOS15, and 35S:MYC-ELF3 for transient expression. The proteins were immunoprecipitated with α-HA antibody and resolved by SDS-PAGE. The immunoblots were probed with α-HA to detect LUX, α-HOS15, or α-MYC to detect ELF3. (D) Gel-filtration analysis of HOS15, LUX, and ELF3. Total proteins extracted from 2-week-old wild type plants were fractionated by size-exclusion chromatography using a Superdex 200 10/300GL column equilibrated with elution buffer [50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 0.02% sodium azide]. The samples were separated by SDS-PAGE and subjected to immunoblotting analysis using α-HOS15, α-LUX, or α-ELF3. Aliquots of total protein extracts from wild type, input after ammonium sulfate precipitation of wild type total extracts, and total protein extracts individually isolated from elf3-1 , hos15-2 , or pcl1-1 mutants were used as input controls. Molecular mass markers (ferritin, 440 kD; β-amylase, 200 kD; alcohol dehydrogenase, 150 kD; BSA, 67 kD) were independently eluted using the same equilibrated column. Total, total protein extract; Input, input after ammonium sulfate precipitation.

    Techniques Used: Bimolecular Fluorescence Complementation Assay, Fluorescence, Confocal Microscopy, In Vivo, Immunoprecipitation, SDS Page, Western Blot, Expressing, Filtration, Size-exclusion Chromatography, Isolation

    29) Product Images from "Pharmacological restoration of autophagy reduces hypertension-related stroke occurrence"

    Article Title: Pharmacological restoration of autophagy reduces hypertension-related stroke occurrence

    Journal: Autophagy

    doi: 10.1080/15548627.2019.1687215

    NMN improved mitochondrial function and cell viability in vitro . ( A ) Evaluation of ultrastructural damage in mitochondria from A10 cells with Ndufc2 knockdown; representative micrographs of mitochondria (left) and graphical representation of the ultrastructural damage in either untreated or treated A10 cells (n = 3). Legend: Nu, nucleus; NM, nuclear membrane, PM, plasma membrane; rER, rough endoplasmic reticulum; Gx, grade of mitochondrial (Mt) damage; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane. (B) Fluorescence microscope analysis of mitochondrial membrane potential (ΔΨm) levels through JC1 dye (n = 3); representative images (left) and corresponding quantification (right) are shown. (C) FACS analysis in A10 cells with Ndufc2 knockdown treated with NaCl without or with NMN (n = 3); CTR indicates non-silenced and untreated cells. Results are presented as mean values ± SEM; *p
    Figure Legend Snippet: NMN improved mitochondrial function and cell viability in vitro . ( A ) Evaluation of ultrastructural damage in mitochondria from A10 cells with Ndufc2 knockdown; representative micrographs of mitochondria (left) and graphical representation of the ultrastructural damage in either untreated or treated A10 cells (n = 3). Legend: Nu, nucleus; NM, nuclear membrane, PM, plasma membrane; rER, rough endoplasmic reticulum; Gx, grade of mitochondrial (Mt) damage; IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane. (B) Fluorescence microscope analysis of mitochondrial membrane potential (ΔΨm) levels through JC1 dye (n = 3); representative images (left) and corresponding quantification (right) are shown. (C) FACS analysis in A10 cells with Ndufc2 knockdown treated with NaCl without or with NMN (n = 3); CTR indicates non-silenced and untreated cells. Results are presented as mean values ± SEM; *p

    Techniques Used: In Vitro, Fluorescence, Microscopy, FACS

    30) Product Images from "Exogenous Pi supplementation improved the salt tolerance of maize (Zea mays L.) by promoting Na+ exclusion"

    Article Title: Exogenous Pi supplementation improved the salt tolerance of maize (Zea mays L.) by promoting Na+ exclusion

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34320-y

    Changes in the length of the primary roots ( A ) and the total number of roots ( B ) of QXN233 under NaCl with/without Pi. The values represent the means ± SEs (n = 3, * P
    Figure Legend Snippet: Changes in the length of the primary roots ( A ) and the total number of roots ( B ) of QXN233 under NaCl with/without Pi. The values represent the means ± SEs (n = 3, * P

    Techniques Used:

    Changes in net Na + and K + fluxes in QXN233. The ion fluxes were measured in the root mature zone of QXN233 under NaCl with/without Pi. The values represent the means ± SEs (n = 3, * P
    Figure Legend Snippet: Changes in net Na + and K + fluxes in QXN233. The ion fluxes were measured in the root mature zone of QXN233 under NaCl with/without Pi. The values represent the means ± SEs (n = 3, * P

    Techniques Used:

    Changes in the Na + ( A ) K + ( B ) and Pi ( D ) contents and the Na + /K + ratio ( C ) in QXN233 under NaCl with/without Pi. The values represent the means ± SEs (n = 3, * P
    Figure Legend Snippet: Changes in the Na + ( A ) K + ( B ) and Pi ( D ) contents and the Na + /K + ratio ( C ) in QXN233 under NaCl with/without Pi. The values represent the means ± SEs (n = 3, * P

    Techniques Used:

    31) Product Images from "Lithium promotes neural precursor cell proliferation: evidence for the involvement of the non-canonical GSK-3?-NF-AT signaling"

    Article Title: Lithium promotes neural precursor cell proliferation: evidence for the involvement of the non-canonical GSK-3?-NF-AT signaling

    Journal: Cell & Bioscience

    doi: 10.1186/2045-3701-1-18

    Calcineurin/NFAT inhibitor cyclosporin A antagonized lithium-induced cell number increase in RG3.6 cell cultures . (A) Lithium did not significantly change β-catenin expression in RG3.6 cells. RG3.6 cells were grown for 3 days in culture medium containing 3 mM LiCl or 3 mM control NaCl. The cells were then lysed in RIPA for western blotting analysis on β-catenin and β-actin expression. (B) Lithium increased nuclear expression of NF-AT. Aliquot of cells from A were used for cytoplamic and nuclear fractionation, followed by western blotting assays for the subcellular expressions of NF-AT. (C) Lithium stimulated transcriptional activation of NF-AT. RG3.6 cells treated with LiCl or NaCl were used for gene reporter assays. The luciferase activity is presented as fold induction relative to that of NaCl-treated cells. *: paired t test, P
    Figure Legend Snippet: Calcineurin/NFAT inhibitor cyclosporin A antagonized lithium-induced cell number increase in RG3.6 cell cultures . (A) Lithium did not significantly change β-catenin expression in RG3.6 cells. RG3.6 cells were grown for 3 days in culture medium containing 3 mM LiCl or 3 mM control NaCl. The cells were then lysed in RIPA for western blotting analysis on β-catenin and β-actin expression. (B) Lithium increased nuclear expression of NF-AT. Aliquot of cells from A were used for cytoplamic and nuclear fractionation, followed by western blotting assays for the subcellular expressions of NF-AT. (C) Lithium stimulated transcriptional activation of NF-AT. RG3.6 cells treated with LiCl or NaCl were used for gene reporter assays. The luciferase activity is presented as fold induction relative to that of NaCl-treated cells. *: paired t test, P

    Techniques Used: Expressing, Western Blot, Fractionation, Activation Assay, Luciferase, Activity Assay

    Lithium had no significant effect on percentage of GFP expressing live cells in RG3.6 cell cultures . (A) GFP-expressing cells are negative for propidium iodide (PI) staining. RG3.6 neurospheres were dissociated by trypsinization. The dissociated cells were stained with PI, a red fluorescent nuclear dye that specifically stain dead cells. The green GFP signal and the red PI staining were visualized and photographed using fluorescent microscope. (B) RG3.6 cells treated with NaCl or LiCl had similar GFP histograms. Flow cytometry analysis was performed on RG3.6 cell cultures treated with 3 mM NaCl or 3 mM LiCl for 3 days. Cells grown in non-FGF2 containing medium for 6 days were used as a negative control to gate GFP signal, since most cells grown in this condition were dead. (C) Quantification of the data shown in B. Lithium treatment had no significant effect on the percentage of GFP-expressing live cells in RG3.6 cell cultures.
    Figure Legend Snippet: Lithium had no significant effect on percentage of GFP expressing live cells in RG3.6 cell cultures . (A) GFP-expressing cells are negative for propidium iodide (PI) staining. RG3.6 neurospheres were dissociated by trypsinization. The dissociated cells were stained with PI, a red fluorescent nuclear dye that specifically stain dead cells. The green GFP signal and the red PI staining were visualized and photographed using fluorescent microscope. (B) RG3.6 cells treated with NaCl or LiCl had similar GFP histograms. Flow cytometry analysis was performed on RG3.6 cell cultures treated with 3 mM NaCl or 3 mM LiCl for 3 days. Cells grown in non-FGF2 containing medium for 6 days were used as a negative control to gate GFP signal, since most cells grown in this condition were dead. (C) Quantification of the data shown in B. Lithium treatment had no significant effect on the percentage of GFP-expressing live cells in RG3.6 cell cultures.

    Techniques Used: Expressing, Staining, Microscopy, Flow Cytometry, Cytometry, Negative Control

    Lithium significantly increased percentage of BrdU + cells in RG3.6 cell cultures . RG3.6 cells were grown in culture medium containing 3 mM LiCl or control NaCl on laminin-coated coverslips for 3 days followed by 4 hours of BrdU labeling and subsequent BrdU immuno-staining. The data represent mean ± standard error. *: Bonferroni/Dunn, P
    Figure Legend Snippet: Lithium significantly increased percentage of BrdU + cells in RG3.6 cell cultures . RG3.6 cells were grown in culture medium containing 3 mM LiCl or control NaCl on laminin-coated coverslips for 3 days followed by 4 hours of BrdU labeling and subsequent BrdU immuno-staining. The data represent mean ± standard error. *: Bonferroni/Dunn, P

    Techniques Used: Labeling, Immunostaining

    Lithium had no significant effect on RNA expression of neurotrophic factors CNTF, GDNF, LIF, NGFβ, NGFγ and NT-3 in rat primary neural precursor cells . Rat primary neural precursor cells were treated with 3 mM LiCl or control NaCl for 3 days. Then the cells were lysed for RNA extraction and subsequent quantitative real time PCR analysis. RNA levels of neurotrophic factors were normalized to peptidylprolyl isomerase A (Ppia). The data represent mean ± standard error. *: Bonferroni/Dunn, P
    Figure Legend Snippet: Lithium had no significant effect on RNA expression of neurotrophic factors CNTF, GDNF, LIF, NGFβ, NGFγ and NT-3 in rat primary neural precursor cells . Rat primary neural precursor cells were treated with 3 mM LiCl or control NaCl for 3 days. Then the cells were lysed for RNA extraction and subsequent quantitative real time PCR analysis. RNA levels of neurotrophic factors were normalized to peptidylprolyl isomerase A (Ppia). The data represent mean ± standard error. *: Bonferroni/Dunn, P

    Techniques Used: RNA Expression, RNA Extraction, Real-time Polymerase Chain Reaction

    Lithium inhibited GSK-3β in RG3.6 cells, and other GSK3β inhibitors mimicked lithium's effect on RG3.6 cell growth . (A) RG3.6 cells were grown in culture medium containing 3 mM LiCl or 3 mM control NaCl for 3 days, followed by western blotting analysis on P-GSK-3β (Ser9) and GSK-3β expression. (B) The blotting results in A were analyzed using ImageJ software ( http://rsb.info.nih.gov/ij/ , 1997-2007), and the ratio of P-GSK-3β was plotted. (C) Other GSK-3β inhibitors, like lithium, also increased cell numbers in RG3.6 cell cultures. Cell count analysis was performed on RG3.6 cells grown for 6 days in culture medium without or with 3 mM LiCl, 0.1% DMSO, 5 μM SB216763, or 25 μM SB415286 (n = 6 for each condition). DMSO was used as the vehicle control for SB216763 and SB415286, since these two drugs were dissolved in DMSO. The data represent mean ± standard error, and significance was determined with Bonferroni/Dunn post hoc analysis following ANOVA.
    Figure Legend Snippet: Lithium inhibited GSK-3β in RG3.6 cells, and other GSK3β inhibitors mimicked lithium's effect on RG3.6 cell growth . (A) RG3.6 cells were grown in culture medium containing 3 mM LiCl or 3 mM control NaCl for 3 days, followed by western blotting analysis on P-GSK-3β (Ser9) and GSK-3β expression. (B) The blotting results in A were analyzed using ImageJ software ( http://rsb.info.nih.gov/ij/ , 1997-2007), and the ratio of P-GSK-3β was plotted. (C) Other GSK-3β inhibitors, like lithium, also increased cell numbers in RG3.6 cell cultures. Cell count analysis was performed on RG3.6 cells grown for 6 days in culture medium without or with 3 mM LiCl, 0.1% DMSO, 5 μM SB216763, or 25 μM SB415286 (n = 6 for each condition). DMSO was used as the vehicle control for SB216763 and SB415286, since these two drugs were dissolved in DMSO. The data represent mean ± standard error, and significance was determined with Bonferroni/Dunn post hoc analysis following ANOVA.

    Techniques Used: Western Blot, Expressing, Software, Cell Counting

    32) Product Images from "Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp"

    Article Title: Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-8-75

    Hlp peptides corresponding to the C-terminal domain bind heparan sulfate . (A) Microtiter-plate wells coated with 50 μL of 0.1 μM Hlp or 50 μL of 0.65 μM individual 30-mer synthetic peptides covering the entire sequence of Hlp were incubated with biotinylated heparan sulfate in phosphate buffer 10 mM, pH 7.2. Heparan sulfate binding was measured by adding streptavidin peroxidase to the wells and expressed in absorbency units at 490 nm. The data are expressed as mean ± SD of a representative experiment from five independent ones performed in duplicate. (B) Microtiter-plate wells were coated with Hlp or peptides p31–60, p136–165, and p151–180 and biotinylated heparan sulfate was added in the presence of increasing concentrations of NaCl. Heparan sulfate binding was quantified as above.
    Figure Legend Snippet: Hlp peptides corresponding to the C-terminal domain bind heparan sulfate . (A) Microtiter-plate wells coated with 50 μL of 0.1 μM Hlp or 50 μL of 0.65 μM individual 30-mer synthetic peptides covering the entire sequence of Hlp were incubated with biotinylated heparan sulfate in phosphate buffer 10 mM, pH 7.2. Heparan sulfate binding was measured by adding streptavidin peroxidase to the wells and expressed in absorbency units at 490 nm. The data are expressed as mean ± SD of a representative experiment from five independent ones performed in duplicate. (B) Microtiter-plate wells were coated with Hlp or peptides p31–60, p136–165, and p151–180 and biotinylated heparan sulfate was added in the presence of increasing concentrations of NaCl. Heparan sulfate binding was quantified as above.

    Techniques Used: Sequencing, Incubation, Binding Assay

    33) Product Images from "Identification and Expression Analysis of a Novel HbCIPK2-Interacting Ferredoxin from Halophyte H. brevisubulatum"

    Article Title: Identification and Expression Analysis of a Novel HbCIPK2-Interacting Ferredoxin from Halophyte H. brevisubulatum

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0144132

    Expression analysis of HbFd1 in H . brevisubulatum by real-time PCR. (A) Expression pattern of HbFd1 from different tissues of H . brevisubulatum . YR, young root; YL, young leaf; steam; YI, young inflorescence; LSh, leaf sheath; ML, mature leaf; MR, mature root and anther. (B) Expression pattern of HbFd1 in H . brevisubulatum under 350 mM NaCl, 350 mM mannitol and 10% PEG6000 stressed for 6 hrs, and 4°C for 12 hrs, respectively. All assays were performed in triplicate. Significant differences were determined relative to each control using a student’s t-test [ P -values
    Figure Legend Snippet: Expression analysis of HbFd1 in H . brevisubulatum by real-time PCR. (A) Expression pattern of HbFd1 from different tissues of H . brevisubulatum . YR, young root; YL, young leaf; steam; YI, young inflorescence; LSh, leaf sheath; ML, mature leaf; MR, mature root and anther. (B) Expression pattern of HbFd1 in H . brevisubulatum under 350 mM NaCl, 350 mM mannitol and 10% PEG6000 stressed for 6 hrs, and 4°C for 12 hrs, respectively. All assays were performed in triplicate. Significant differences were determined relative to each control using a student’s t-test [ P -values

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Serial Time-encoded Amplified Microscopy

    34) Product Images from "Structure of the Peptidoglycan Synthase Activator LpoP in Pseudomonas aeruginosa"

    Article Title: Structure of the Peptidoglycan Synthase Activator LpoP in Pseudomonas aeruginosa

    Journal: Structure(London, England:1993)

    doi: 10.1016/j.str.2020.03.012

    Interaction of LpoP with the Pa UB2H Domain of Pa PBP1B (A) Quantified BLI binding data for biotin-labeled Pa UB2H binding to LpoP. (B) Region of 1 H– 15 N correlation spectra showing chemical shift perturbations induced on 15 N-labeled LpoP by addition of different ratios of Pa UB2H. The spectra plotted in black, blue, and red correspond to a UB2H/LpoP ratio of 0, 1.2, and 1.8, respectively. The samples were prepared with 115 μM of 15 N LpoP in 50 mM HEPES buffer (pH 7.0) containing 150 mM NaCl. Pa UB2H was concentrated in the exact same buffer to reach a final concentration of 150 μM and added to the NMR tube to obtain the different ratios. The NMR experiments were recorded at 25°C and on a 20-T NMR spectrometer. (C) Residues showing a higher perturbation than 0.015 ppm upon UB2H addition are mapped in red on the surface and cartoon representation of the LpoP structure. (D) Sequence conservation scores were calculated using the ConSurf webserver ( Landau et al., 2005 ) and displayed on the surface representation of LpoP using the same orientation as (C). Scores range from 1 (not conserved, cyan) to 9 (highly conserved, magenta).
    Figure Legend Snippet: Interaction of LpoP with the Pa UB2H Domain of Pa PBP1B (A) Quantified BLI binding data for biotin-labeled Pa UB2H binding to LpoP. (B) Region of 1 H– 15 N correlation spectra showing chemical shift perturbations induced on 15 N-labeled LpoP by addition of different ratios of Pa UB2H. The spectra plotted in black, blue, and red correspond to a UB2H/LpoP ratio of 0, 1.2, and 1.8, respectively. The samples were prepared with 115 μM of 15 N LpoP in 50 mM HEPES buffer (pH 7.0) containing 150 mM NaCl. Pa UB2H was concentrated in the exact same buffer to reach a final concentration of 150 μM and added to the NMR tube to obtain the different ratios. The NMR experiments were recorded at 25°C and on a 20-T NMR spectrometer. (C) Residues showing a higher perturbation than 0.015 ppm upon UB2H addition are mapped in red on the surface and cartoon representation of the LpoP structure. (D) Sequence conservation scores were calculated using the ConSurf webserver ( Landau et al., 2005 ) and displayed on the surface representation of LpoP using the same orientation as (C). Scores range from 1 (not conserved, cyan) to 9 (highly conserved, magenta).

    Techniques Used: Binding Assay, Labeling, Concentration Assay, Nuclear Magnetic Resonance, Sequencing

    35) Product Images from "ITC Analysis of Ligand Binding to PreQ1 Riboswitches"

    Article Title: ITC Analysis of Ligand Binding to PreQ1 Riboswitches

    Journal: Methods in enzymology

    doi: 10.1016/B978-0-12-801122-5.00018-0

    Representative isotherm and binding-model fit for a preQ 1 -I type III riboswitch. ITC was performed at 25 °C in 0.0060 M MgCl 2 , 0.10 M NaCl, and 0.050 M Na-HEPES pH 7.0. The wild type riboswitch was in the cell at 8.74 μM; preQ 1 was in the syringe at a concentration 10-fold higher than the RNA. The c value is 112. The parameters obtained from a “One Set of Sites” binding model are shown as text in the inset DeltaH window. The values are: the binding stoichiometry, N = 0.93; K A = 1.39 x 10 7 M −1 ; Δ H = −25.39 x 10 3 cal/mol; and ΔS = −52.4 cal/(mol K).
    Figure Legend Snippet: Representative isotherm and binding-model fit for a preQ 1 -I type III riboswitch. ITC was performed at 25 °C in 0.0060 M MgCl 2 , 0.10 M NaCl, and 0.050 M Na-HEPES pH 7.0. The wild type riboswitch was in the cell at 8.74 μM; preQ 1 was in the syringe at a concentration 10-fold higher than the RNA. The c value is 112. The parameters obtained from a “One Set of Sites” binding model are shown as text in the inset DeltaH window. The values are: the binding stoichiometry, N = 0.93; K A = 1.39 x 10 7 M −1 ; Δ H = −25.39 x 10 3 cal/mol; and ΔS = −52.4 cal/(mol K).

    Techniques Used: Binding Assay, Concentration Assay

    36) Product Images from "ATM, a DNA damage-inducible kinase, contributes to activation by high NaCl of the transcription factor TonEBP/OREBP"

    Article Title: ATM, a DNA damage-inducible kinase, contributes to activation by high NaCl of the transcription factor TonEBP/OREBP

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

    doi: 10.1073/pnas.0403062101

    ATM is activated by different DNA damaging agents, including high NaCl, but only high NaCl increases transcriptional activity of TonEBP/OREBP. ( A ) Western blot analysis of HEK293 cells by using anti-ATM or phosphospecific anti-ATM 1981S-P. Cells were
    Figure Legend Snippet: ATM is activated by different DNA damaging agents, including high NaCl, but only high NaCl increases transcriptional activity of TonEBP/OREBP. ( A ) Western blot analysis of HEK293 cells by using anti-ATM or phosphospecific anti-ATM 1981S-P. Cells were

    Techniques Used: Activity Assay, Western Blot

    Wortmannin inhibits TonEBP/OREBP transcriptional activity and BGT1 mRNA abundance when NaCl is high. HEK293 cells ( A ) or AT cells ( B ) were preincubated with wortmannin for 1 h at 300 mosmol/kg before increasing osmolality to 500 mosmol/kg by adding NaCl.
    Figure Legend Snippet: Wortmannin inhibits TonEBP/OREBP transcriptional activity and BGT1 mRNA abundance when NaCl is high. HEK293 cells ( A ) or AT cells ( B ) were preincubated with wortmannin for 1 h at 300 mosmol/kg before increasing osmolality to 500 mosmol/kg by adding NaCl.

    Techniques Used: Activity Assay

    37) Product Images from "The Cryptococcus neoformans Alkaline Response Pathway: Identification of a Novel Rim Pathway Activator"

    Article Title: The Cryptococcus neoformans Alkaline Response Pathway: Identification of a Novel Rim Pathway Activator

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005159

    Rim101 proteolysis and nuclear localization are dependent on pH. (A) GFP-Rim101 is proteolytically processed from 140 kDa to ~100 kDa in response to increasing pH. GFP-Rim101 was immunoprecipitated from wild-type cells after incubating for 5 hr at the indicated pH 8 (SC medium buffered with McIlvaine’s buffer). Protein processing was determined by western blotting using an α-GFP antibody. (B) GFP-Rim101 nuclear localization increases in response to increasing pH. Cells were cultured in the same way as in (A). GFP signal was assessed by epifluorescence microscopy. Nuclei were stained using Hoechst 33342 live nuclei stain. Scale bar = 5 μm. (C) GFP-Rim101 proteolysis is not induced by 1 M NaCl or 150 μM BPS. Cells were cultured in each indicated condition for 3 hr. GFP-Rim101 was analyzed by western blot. (D) GFP-Rim101 localization in response to pH 7 SC (McIlvaine’s) or pH 4 SC (McIlvaine’s) with 1 M NaCl or 150 μM BPS. Cell assessed epifluorescence microscopy after 30 min incubation in each condition. Scale bar = 5 μm (E) rim101Δ is not NaCl sensitive at pH 4. Strains spotted onto YPD, YPD 150mM HEPES pH 4 1.5 M NaCl, and YPD 1.5 M NaCl.
    Figure Legend Snippet: Rim101 proteolysis and nuclear localization are dependent on pH. (A) GFP-Rim101 is proteolytically processed from 140 kDa to ~100 kDa in response to increasing pH. GFP-Rim101 was immunoprecipitated from wild-type cells after incubating for 5 hr at the indicated pH 8 (SC medium buffered with McIlvaine’s buffer). Protein processing was determined by western blotting using an α-GFP antibody. (B) GFP-Rim101 nuclear localization increases in response to increasing pH. Cells were cultured in the same way as in (A). GFP signal was assessed by epifluorescence microscopy. Nuclei were stained using Hoechst 33342 live nuclei stain. Scale bar = 5 μm. (C) GFP-Rim101 proteolysis is not induced by 1 M NaCl or 150 μM BPS. Cells were cultured in each indicated condition for 3 hr. GFP-Rim101 was analyzed by western blot. (D) GFP-Rim101 localization in response to pH 7 SC (McIlvaine’s) or pH 4 SC (McIlvaine’s) with 1 M NaCl or 150 μM BPS. Cell assessed epifluorescence microscopy after 30 min incubation in each condition. Scale bar = 5 μm (E) rim101Δ is not NaCl sensitive at pH 4. Strains spotted onto YPD, YPD 150mM HEPES pH 4 1.5 M NaCl, and YPD 1.5 M NaCl.

    Techniques Used: Immunoprecipitation, Western Blot, Cell Culture, Epifluorescence Microscopy, Staining, Incubation

    The rra1Δ mutant is phenotypically identical to other Rim pathway mutants. (A) rra1Δ insertional mutant has pH 8 and 1.5 M NaCl growth defects that are rescued by GFP-RIM101T expression. 10-fold serial dilutions were spotted onto YPD, YPD with 150 mM HEPES pH 8, and YPD with 1.5 M NaCl. (B) Independent rra1Δ mutant has a growth defect pH 8 and 1.5 M NaCl. (C) The rra1Δ strain has a capsule defect. Cells were cultured for 48 hr in CO 2 -independent media at 37 ° C to induce capsule. Capsule was visualized by India ink staining. Scale bar = 5 μm. (D) The rra1Δ mutation disrupts GFP-Rim101 proteolysis. GFP-Rim101 was immunoprecipitated from the indicated mutant strains after 5 hr incubation in YPD with 150 mM HEPES at pH 7.4. (E) GFP-Rim101 nuclear localization is disrupted in the rra1Δ mutant. GFP-Rim101 was assessed after 5 hr incubation in pH 8 SC McIlvaine’s buffer.
    Figure Legend Snippet: The rra1Δ mutant is phenotypically identical to other Rim pathway mutants. (A) rra1Δ insertional mutant has pH 8 and 1.5 M NaCl growth defects that are rescued by GFP-RIM101T expression. 10-fold serial dilutions were spotted onto YPD, YPD with 150 mM HEPES pH 8, and YPD with 1.5 M NaCl. (B) Independent rra1Δ mutant has a growth defect pH 8 and 1.5 M NaCl. (C) The rra1Δ strain has a capsule defect. Cells were cultured for 48 hr in CO 2 -independent media at 37 ° C to induce capsule. Capsule was visualized by India ink staining. Scale bar = 5 μm. (D) The rra1Δ mutation disrupts GFP-Rim101 proteolysis. GFP-Rim101 was immunoprecipitated from the indicated mutant strains after 5 hr incubation in YPD with 150 mM HEPES at pH 7.4. (E) GFP-Rim101 nuclear localization is disrupted in the rra1Δ mutant. GFP-Rim101 was assessed after 5 hr incubation in pH 8 SC McIlvaine’s buffer.

    Techniques Used: Mutagenesis, Expressing, Cell Culture, Staining, Immunoprecipitation, Incubation

    Role of Rim13 and Rim23 orthologs in Rim101-regulated phenotypes. (A) The C . neoformans RIM13 and RIM23 orthologs are required for pH 8 and NaCl tolerance. 10-fold serial dilutions of the indicated strains were spotted onto YPD, YPD 150mM HEPES pH 8, YPD 1.5M NaCl and incubated at 30 ° C for 48 hr -72 hr (B) The rim13Δ and rim23Δ mutants have a rim101Δ- like capsule defect. Cells were incubated in CO 2 -independent media for 48hr at 37 ° C. Capsule was visualized by counterstaining with India ink. (C) Rim101 proteolysis and localization are disrupted in rim13Δ , rim20Δ , and rim23Δ mutant strains. GFP-Rim101 was immunoprecipitated from each strain after 5 hr incubation in pH 7.4 YPD buffered with 150 mM HEPES. (D) GFP-Rim101 localization was assessed in the indicated strains after culturing for 5 hr in SC medium buffered with McIlvaine’s buffered to pH 8. Nuclei were stained with Hoechst 33342 live nuclei stain. Scale bar = 5 μm.
    Figure Legend Snippet: Role of Rim13 and Rim23 orthologs in Rim101-regulated phenotypes. (A) The C . neoformans RIM13 and RIM23 orthologs are required for pH 8 and NaCl tolerance. 10-fold serial dilutions of the indicated strains were spotted onto YPD, YPD 150mM HEPES pH 8, YPD 1.5M NaCl and incubated at 30 ° C for 48 hr -72 hr (B) The rim13Δ and rim23Δ mutants have a rim101Δ- like capsule defect. Cells were incubated in CO 2 -independent media for 48hr at 37 ° C. Capsule was visualized by counterstaining with India ink. (C) Rim101 proteolysis and localization are disrupted in rim13Δ , rim20Δ , and rim23Δ mutant strains. GFP-Rim101 was immunoprecipitated from each strain after 5 hr incubation in pH 7.4 YPD buffered with 150 mM HEPES. (D) GFP-Rim101 localization was assessed in the indicated strains after culturing for 5 hr in SC medium buffered with McIlvaine’s buffered to pH 8. Nuclei were stained with Hoechst 33342 live nuclei stain. Scale bar = 5 μm.

    Techniques Used: Incubation, Mutagenesis, Immunoprecipitation, Staining

    ESCRT complex proteins, Vps23 and Snf7, are required for Rim101 activation. (A) snf7Δ and vps23Δ capsule defects are partially rescued by GFP-RIM101T expression. Strains cultured for 24 hr in tissue culture media. India ink used to visualize capsule. (B) GFP-RIM101T expression rescues the vps23Δ and snf7Δ growth defects on pH 8 but not 1.5 M NaCl. (C) GFP-Rim101 was immunoprecipitated from the indicated strains after 5 hr incubation in YPD with 150mM HEPES at pH 7.4. (D) GFP-Rim101 (full-length) nuclear localization is disrupted in the vps23Δ mutant. Localization was assessed after culturing for 5 hr in SC with McIlvaine’s buffer at pH 8. Nuclei were stained with Hoechst 33342 live nuclear stain. Scale bar = 5 μm.
    Figure Legend Snippet: ESCRT complex proteins, Vps23 and Snf7, are required for Rim101 activation. (A) snf7Δ and vps23Δ capsule defects are partially rescued by GFP-RIM101T expression. Strains cultured for 24 hr in tissue culture media. India ink used to visualize capsule. (B) GFP-RIM101T expression rescues the vps23Δ and snf7Δ growth defects on pH 8 but not 1.5 M NaCl. (C) GFP-Rim101 was immunoprecipitated from the indicated strains after 5 hr incubation in YPD with 150mM HEPES at pH 7.4. (D) GFP-Rim101 (full-length) nuclear localization is disrupted in the vps23Δ mutant. Localization was assessed after culturing for 5 hr in SC with McIlvaine’s buffer at pH 8. Nuclei were stained with Hoechst 33342 live nuclear stain. Scale bar = 5 μm.

    Techniques Used: Activation Assay, Expressing, Cell Culture, Immunoprecipitation, Incubation, Mutagenesis, Staining

    38) Product Images from "Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease"

    Article Title: Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0165463

    2–6 dithiopurine stabilizes human lysosomal alpha-galactosidase in thermal shift assay. Panel A. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was equilibrated in the presence of ligands dissolved in DMSO 20%: DGJ 1 microM (empty triangle) or 40 microM (empty circle); DTP 6 mM (empty diamond); DTP 6 mM plus DGJ 1 microM (filled circle); DTP 6 mM plus DGJ 40 microM (filled triangle). A control (with only DMSO) was also shown (x). Panel B. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was incubated in the presence of DTP 6 mM for 1 hour at 4°C then DTP was eliminated by dialysis. A control experiment was conducted by incubating the enzyme only with DMSO. Both the aliquots of Fabrazyme ® were analysed by thermal shift assay in the presence of DTP 6 mM or in the presence of DMSO. Filled squares: DTP/DTP; open squares: DMSO/DTP; filled circles: DTP/DMSO; open circles: DMSO/DMSO where the first word of the label corresponds to the pretreatment and the second part corresponds to the presence of the compound during the thermal scan. The protein samples were heated from 20 to 90° at 1°C/min in the presence of Sypro Orange 2.5x. Data were shown as normalized curves.
    Figure Legend Snippet: 2–6 dithiopurine stabilizes human lysosomal alpha-galactosidase in thermal shift assay. Panel A. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was equilibrated in the presence of ligands dissolved in DMSO 20%: DGJ 1 microM (empty triangle) or 40 microM (empty circle); DTP 6 mM (empty diamond); DTP 6 mM plus DGJ 1 microM (filled circle); DTP 6 mM plus DGJ 40 microM (filled triangle). A control (with only DMSO) was also shown (x). Panel B. Fabrazyme ® (in Na-Hepes 20 mM, NaCl 150 mM, pH 7.4) was incubated in the presence of DTP 6 mM for 1 hour at 4°C then DTP was eliminated by dialysis. A control experiment was conducted by incubating the enzyme only with DMSO. Both the aliquots of Fabrazyme ® were analysed by thermal shift assay in the presence of DTP 6 mM or in the presence of DMSO. Filled squares: DTP/DTP; open squares: DMSO/DTP; filled circles: DTP/DMSO; open circles: DMSO/DMSO where the first word of the label corresponds to the pretreatment and the second part corresponds to the presence of the compound during the thermal scan. The protein samples were heated from 20 to 90° at 1°C/min in the presence of Sypro Orange 2.5x. Data were shown as normalized curves.

    Techniques Used: Thermal Shift Assay, Incubation

    39) Product Images from "The Arabidopsis Ca2+-Dependent Protein Kinase CPK12 Is Involved in Plant Response to Salt Stress"

    Article Title: The Arabidopsis Ca2+-Dependent Protein Kinase CPK12 Is Involved in Plant Response to Salt Stress

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19124062

    Effect of NaCl on activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in wild-type (GL1) and CPK12 -RNAi lines. Seven-day-old seedlings were transferred to MS medium supplemented with or without 100 mM NaCl for 10 d. The activities of antioxidant enzymes were analyzed. Each column shows the mean of three replicated experiments and bars represent the standard error of the mean. Columns labeled with letters in the same group denote significant difference at p
    Figure Legend Snippet: Effect of NaCl on activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in wild-type (GL1) and CPK12 -RNAi lines. Seven-day-old seedlings were transferred to MS medium supplemented with or without 100 mM NaCl for 10 d. The activities of antioxidant enzymes were analyzed. Each column shows the mean of three replicated experiments and bars represent the standard error of the mean. Columns labeled with letters in the same group denote significant difference at p

    Techniques Used: Mass Spectrometry, Labeling

    Effect of salt stress on cell viability in wild-type (GL1) and CPK12 -RNAi lines. ( A ) Seven-day-old seedlings were transferred to MS medium supplemented with or without 100 mM NaCl for 12 h. Cell viability was assayed with fluorescein diacetate (FDA, green) stain. Representative images of apical region of roots are shown. Scale bars, 100 μm. ( B ) The fluorescence intensity (±SD) represents the mean of 10 independent seedlings. The mean values of FDA fluorescence are labeled with letters in the same group to denote significant differences ( p
    Figure Legend Snippet: Effect of salt stress on cell viability in wild-type (GL1) and CPK12 -RNAi lines. ( A ) Seven-day-old seedlings were transferred to MS medium supplemented with or without 100 mM NaCl for 12 h. Cell viability was assayed with fluorescein diacetate (FDA, green) stain. Representative images of apical region of roots are shown. Scale bars, 100 μm. ( B ) The fluorescence intensity (±SD) represents the mean of 10 independent seedlings. The mean values of FDA fluorescence are labeled with letters in the same group to denote significant differences ( p

    Techniques Used: Mass Spectrometry, Staining, Fluorescence, Labeling

    Changes in CPK12 expression alter expression of a subset of genes involved in salt stress responses. The mRNA levels in the seedling of wild-type GL1, CPK12 -RNAi mutants were determined by qRT-PCR. One-week-old seedlings were transferred to MS medium with and without the addition of 100 mM NaCl for ten days. The expression of salt stress responsive genes was analyzed. The gene expression levels were normalized relative to the value of the GL1 plants. Each value is the mean of the three independent determinations; columns labeled with letters in the same group indicate significant differences ( p
    Figure Legend Snippet: Changes in CPK12 expression alter expression of a subset of genes involved in salt stress responses. The mRNA levels in the seedling of wild-type GL1, CPK12 -RNAi mutants were determined by qRT-PCR. One-week-old seedlings were transferred to MS medium with and without the addition of 100 mM NaCl for ten days. The expression of salt stress responsive genes was analyzed. The gene expression levels were normalized relative to the value of the GL1 plants. Each value is the mean of the three independent determinations; columns labeled with letters in the same group indicate significant differences ( p

    Techniques Used: Expressing, Quantitative RT-PCR, Mass Spectrometry, Labeling

    Na + levels in root cells of wild-type (WT) GL1 and CPK12 -RNAi plants under salt stress. ( A ) Seven-day-old seedlings were transferred to MS medium supplemented with (120 mM, 150 mM) or without NaCl (0 mM) for 12 h, then seedlings were treated with CoroNa-Green AM (green fluorescence, sodium-specific) for 1 h. Green fluorescence in root cells was observed at the apical region of roots using a Leica confocal microscope. Typical images show Na + content in plant roots. Scale bars, 100 μm. ( B ) The mean relative fluorescence values marked with letters in the same group represent significant differences ( p
    Figure Legend Snippet: Na + levels in root cells of wild-type (WT) GL1 and CPK12 -RNAi plants under salt stress. ( A ) Seven-day-old seedlings were transferred to MS medium supplemented with (120 mM, 150 mM) or without NaCl (0 mM) for 12 h, then seedlings were treated with CoroNa-Green AM (green fluorescence, sodium-specific) for 1 h. Green fluorescence in root cells was observed at the apical region of roots using a Leica confocal microscope. Typical images show Na + content in plant roots. Scale bars, 100 μm. ( B ) The mean relative fluorescence values marked with letters in the same group represent significant differences ( p

    Techniques Used: Mass Spectrometry, Fluorescence, Microscopy

    Ca 2+ levels within roots of GL1 and CPK12 -RNAi plants. ( A ) Seven-day-old seedlings were transferred to MS medium supplemented with (100 mM) or without NaCl (0 mM) for 12 h, then stained with the Ca 2+ -specific fluorescent probe Rhod-2 AM for 1 h at room temperature. Orange-red fluorescence within cells was detected at the apical region of roots under a Leica confocal microscope. Representative confocal images show cytosolic Ca 2+ content in plant roots. Scale bars, 100 μm. ( B ) The relative fluorescence intensity (±SD) represents the mean of 10 independent seedlings. The mean values of Ca 2+ fluorescence are labeled with letters in the same group to denote significant differences ( p
    Figure Legend Snippet: Ca 2+ levels within roots of GL1 and CPK12 -RNAi plants. ( A ) Seven-day-old seedlings were transferred to MS medium supplemented with (100 mM) or without NaCl (0 mM) for 12 h, then stained with the Ca 2+ -specific fluorescent probe Rhod-2 AM for 1 h at room temperature. Orange-red fluorescence within cells was detected at the apical region of roots under a Leica confocal microscope. Representative confocal images show cytosolic Ca 2+ content in plant roots. Scale bars, 100 μm. ( B ) The relative fluorescence intensity (±SD) represents the mean of 10 independent seedlings. The mean values of Ca 2+ fluorescence are labeled with letters in the same group to denote significant differences ( p

    Techniques Used: Mass Spectrometry, Staining, Fluorescence, Microscopy, Labeling

    Na + flux in GL1 and CPK12 -RNAi plants. Seeds were germinated for one week in a vertical direction on MS agar medium containing 0, 110, 120, 130 mM NaCl. Continuous NMT recording were applied at the meristem region of the root tips. Each column is the mean of six independent seedlings; bars show the standard error of the mean. Columns marked with letters in the same group indicate significant differences at p
    Figure Legend Snippet: Na + flux in GL1 and CPK12 -RNAi plants. Seeds were germinated for one week in a vertical direction on MS agar medium containing 0, 110, 120, 130 mM NaCl. Continuous NMT recording were applied at the meristem region of the root tips. Each column is the mean of six independent seedlings; bars show the standard error of the mean. Columns marked with letters in the same group indicate significant differences at p

    Techniques Used: Mass Spectrometry

    Accumulation of H 2 O 2 in the root tips of GL1 and CPK12 -RNAi plants exposed to salt stress. ( A ) After germinating seven days, the Arabidopsis seedlings were transferred to MS medium containing 0 or 100 mM NaCl for 10 min, 12 h, or 24 h. These seedlings were incubated with H 2 DCF-DA for 5 min. The green fluorescence within cells at the apical region of roots was detected using a Leica confocal microscope. Scale bars, 100 μm. ( B ) The relative fluorescence intensity (±SD) represents the mean of 10 Arabidopsis seedlings. The mean values of H 2 O 2 fluorescence are labeled with letters in the same group to denote significant differences ( p
    Figure Legend Snippet: Accumulation of H 2 O 2 in the root tips of GL1 and CPK12 -RNAi plants exposed to salt stress. ( A ) After germinating seven days, the Arabidopsis seedlings were transferred to MS medium containing 0 or 100 mM NaCl for 10 min, 12 h, or 24 h. These seedlings were incubated with H 2 DCF-DA for 5 min. The green fluorescence within cells at the apical region of roots was detected using a Leica confocal microscope. Scale bars, 100 μm. ( B ) The relative fluorescence intensity (±SD) represents the mean of 10 Arabidopsis seedlings. The mean values of H 2 O 2 fluorescence are labeled with letters in the same group to denote significant differences ( p

    Techniques Used: Mass Spectrometry, Incubation, Fluorescence, Microscopy, Labeling

    40) Product Images from "Divalent cation-induced conformational changes of influenza virus hemagglutinin"

    Article Title: Divalent cation-induced conformational changes of influenza virus hemagglutinin

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-72368-x

    Characterization of HA proteins in the presence of divalent cations. ( A ) Native polyacrylamide gel electrophoresis results showing the band shifts in the presence of Zn 2+ and Cu 2+ (upper panel). The CU44 HA protein was isolated as a trimer, whereas the two cations induced multimerization or aggregation of the HA molecules. Differential scanning fluorimetry transition curves of HA proteins at different divalent cations (lower panel). HA protein was incubated at 25 °C, and then the temperature was increased by 0.5 °C every 30 s for 50 min. ( B ) The shift in elution volume of the CU44 HA in the presence of Zn 2+ (upper panel) and acidic pH conditions (lower panel) in SEC. The experiment was performed using a Superdex 200 GL column equilibrated with 50 mM Tris (pH 8.0) and 100 mM NaCl, and the shifts were monitored as the concentration of Zn 2+ increased from 0.1 to 1.0 mM. ( C ) The SEC-MALS results of CU44 HA. The HA was incubated with 1 mM Zn 2+ or 100 mM MES (pH 5.5) at 24 °C for 30 min and analyzed using SEC on a Superdex 200 GL column equilibrated with PBS at a flow rate of 0.5 ml/min. ( D ) Antibody recognition of HA conformations, CR9114 (upper panel) and D2 H1-1/H3-1 (lower panel). CU44 HA was incubated with 1 mM Zn 2+ or 100 mM MES (pH 5.5) for ELISA at 4 °C overnight. The stem-specific CR9114 or head-specific D2 H1-1/H3-1 at concentrations of 400, 80, 1, 3, 0.64, 0.128, and 0.026 ng per well was used and detected by anti-human IgG1-HRP as the secondary antibody. Absorbance at 450 nm was monitored.
    Figure Legend Snippet: Characterization of HA proteins in the presence of divalent cations. ( A ) Native polyacrylamide gel electrophoresis results showing the band shifts in the presence of Zn 2+ and Cu 2+ (upper panel). The CU44 HA protein was isolated as a trimer, whereas the two cations induced multimerization or aggregation of the HA molecules. Differential scanning fluorimetry transition curves of HA proteins at different divalent cations (lower panel). HA protein was incubated at 25 °C, and then the temperature was increased by 0.5 °C every 30 s for 50 min. ( B ) The shift in elution volume of the CU44 HA in the presence of Zn 2+ (upper panel) and acidic pH conditions (lower panel) in SEC. The experiment was performed using a Superdex 200 GL column equilibrated with 50 mM Tris (pH 8.0) and 100 mM NaCl, and the shifts were monitored as the concentration of Zn 2+ increased from 0.1 to 1.0 mM. ( C ) The SEC-MALS results of CU44 HA. The HA was incubated with 1 mM Zn 2+ or 100 mM MES (pH 5.5) at 24 °C for 30 min and analyzed using SEC on a Superdex 200 GL column equilibrated with PBS at a flow rate of 0.5 ml/min. ( D ) Antibody recognition of HA conformations, CR9114 (upper panel) and D2 H1-1/H3-1 (lower panel). CU44 HA was incubated with 1 mM Zn 2+ or 100 mM MES (pH 5.5) for ELISA at 4 °C overnight. The stem-specific CR9114 or head-specific D2 H1-1/H3-1 at concentrations of 400, 80, 1, 3, 0.64, 0.128, and 0.026 ng per well was used and detected by anti-human IgG1-HRP as the secondary antibody. Absorbance at 450 nm was monitored.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Isolation, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

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    Article Title: Tau protein liquid–liquid phase separation can initiate tau aggregation
    Article Snippet: .. Excess dye was removed by dialysis (SlideLizer MWCO 10 kDa, Pierce) against (10 mM HEPES buffer, 20 mM NaCl, pH 7.4) at 4°C overnight. p‐tau441‐a568 was produced by labeling p‐tau441 using thiol‐reactive AlexaFluor568 C5 maleimide (Life Technologies) according to the manual. .. The labeling reaction was carried out at RT for 3 h, and excess dye was removed with a NAP‐5 column (GE Healthcare).

    Protease Inhibitor:

    Article Title: Characterization of the Interactions between Calmodulin and Death Receptor 5 in Triple-negative and Estrogen Receptor-positive Breast Cancer Cells
    Article Snippet: .. ER-positive or triple-negative breast cancer cells were lysed using a 50 m m Tris-HCL pH 7.5, 150 m m NaCl, 1% Triton X-100, and 1:100 Halt protease inhibitor (Thermo Fisher Scientific, Waltham, MA) buffer at 4 °C for 30 min. .. The total protein concentration was determined using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific).

    Incubation:

    Article Title: A monovalent ion in the DNA binding interface of the eukaryotic junction-resolving enzyme GEN1
    Article Snippet: .. 8 μl of 0.2 –1 nM radioactively [5′-32 P]-labeled DNA in 10 mM Tris (pH7.5), 0.1% BSA and 100 mM KCl or NaCl were mixed with serial dilutions of 8 μl of Ct GEN1 in 10 mM Tris (pH 7.5), 100 mM NDSB-195 (Affymetrix), 0.1% BSA and 1 mM EDTA and incubated for 20 min at 20°C. .. After the addition of Ficoll-400 to 2.5%, the samples were loaded onto a 6 or 10% (29:1) polyacrylamide gel in TBE and subjected to electrophoresis for 1.5–2.5 h. Gels were dried onto Whatman 3MM paper and free and bound DNA quantified by autoradiography.

    Article Title: Release of Periplasmic Proteins of Brucella suis upon Acidic Shock Involves the Outer Membrane Protein Omp25
    Article Snippet: .. The resulting pellets were resuspended in 400 ml of 25 mM ammonium acetate (pH 4) containing 120 mM NaCl or other buffers (PBS or RPMI 1640 [Life Technology], minimal medium A [ ], or 25 mM sodium citrate [pH 4] containing 120 mM NaCl) and incubated at 37°C for 150 min with shaking. .. After centrifugation at 9,500 × g for 15 min at 4°C, the supernatant (supernatant 1 [S1]; 400 ml) was filtered through a 0.22-μm-pore-size filter (Steritop; Millipore) and concentrated to 20 ml (S2) by ultrafiltration (Amicon concentration cell) through a 10-kDa-cutoff membrane.

    Labeling:

    Article Title: Tau protein liquid–liquid phase separation can initiate tau aggregation
    Article Snippet: .. Excess dye was removed by dialysis (SlideLizer MWCO 10 kDa, Pierce) against (10 mM HEPES buffer, 20 mM NaCl, pH 7.4) at 4°C overnight. p‐tau441‐a568 was produced by labeling p‐tau441 using thiol‐reactive AlexaFluor568 C5 maleimide (Life Technologies) according to the manual. .. The labeling reaction was carried out at RT for 3 h, and excess dye was removed with a NAP‐5 column (GE Healthcare).

    Staining:

    Article Title: Novel Hepatitis B Virus Capsid-Targeting Antiviral that Aggregates Core Particles and Inhibits Nuclear Entry of Viral Cores
    Article Snippet: .. In a final reaction volume of 20 μl, 10 μl of Cp (15 μM dimer) in Buffer N was mixed with 10 μl assembly buffer [100 mM HEPES (pH 7.5), 1 M NaCl] containing 2x SYPRO Orange Protein Gel Stain (Life Technologies). .. Compounds were added at a final concentration of 20 μM, and reactions contained 1% DMSO.

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    Thermo Fisher sodium chloride nacl
    Spearman correlation between salt ST and salt PR. Spearman correlation was used to determine the correlation between salt ST and <t>umami</t> PR. The variables were logged to reduce the magnitude of their scales. Salt ST was measured by the AUC of intensity ratings for <t>NaCl</t> and salt PR was determined using the Monell forced-choice, paired-comparison tracking method for NaCl. Salt ST and PR were negatively correlated with a Spearman correlation coefficient of –0.35 ( P
    Sodium Chloride Nacl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Scientific Remel Lowenstein Jensen Medium with 5 NaCl LJ is for the detection of sodium chloride tolerance to aid in the differentiation of Mycobacterium spp
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    Spearman correlation between salt ST and salt PR. Spearman correlation was used to determine the correlation between salt ST and umami PR. The variables were logged to reduce the magnitude of their scales. Salt ST was measured by the AUC of intensity ratings for NaCl and salt PR was determined using the Monell forced-choice, paired-comparison tracking method for NaCl. Salt ST and PR were negatively correlated with a Spearman correlation coefficient of –0.35 ( P

    Journal: Chemical Senses

    Article Title: Taste Sensitivity and Taste Preference Measures Are Correlated in Healthy Young Adults

    doi: 10.1093/chemse/bjy082

    Figure Lengend Snippet: Spearman correlation between salt ST and salt PR. Spearman correlation was used to determine the correlation between salt ST and umami PR. The variables were logged to reduce the magnitude of their scales. Salt ST was measured by the AUC of intensity ratings for NaCl and salt PR was determined using the Monell forced-choice, paired-comparison tracking method for NaCl. Salt ST and PR were negatively correlated with a Spearman correlation coefficient of –0.35 ( P

    Article Snippet: The tastants were: sucrose for sweet taste (Thermo Fisher; S5-500), monosodium glutamate (MSG) for umami taste (Thermo Fisher; ICN10180080), inosine monophosphate (IMP) for umami taste (Thermo Fisher; AC226260250), MSG + IMP, sodium chloride (NaCl) for salt taste (Thermo Fisher; S641-500), citric acid for sour taste (A940-500), [...] and oleic acid for fat taste [...] (A195-500; Thermo Fisher Scientific).

    Techniques:

    CaM directly interacts with DR5 in DR5 death domain in a Ca 2+ -dependent manner. A , CaM pull-down of purified DR5 cytoplasmic region (DR5 CR) and DR5 death domain (DR5 DD) in a 50 m m Tris, pH 7.6, 120 m m NaCl, 1% Brij buffer with 1 m m Ca 2+ , or 2 m m EGTA.

    Journal: The Journal of Biological Chemistry

    Article Title: Characterization of the Interactions between Calmodulin and Death Receptor 5 in Triple-negative and Estrogen Receptor-positive Breast Cancer Cells

    doi: 10.1074/jbc.M116.727727

    Figure Lengend Snippet: CaM directly interacts with DR5 in DR5 death domain in a Ca 2+ -dependent manner. A , CaM pull-down of purified DR5 cytoplasmic region (DR5 CR) and DR5 death domain (DR5 DD) in a 50 m m Tris, pH 7.6, 120 m m NaCl, 1% Brij buffer with 1 m m Ca 2+ , or 2 m m EGTA.

    Article Snippet: ER-positive or triple-negative breast cancer cells were lysed using a 50 m m Tris-HCL pH 7.5, 150 m m NaCl, 1% Triton X-100, and 1:100 Halt protease inhibitor (Thermo Fisher Scientific, Waltham, MA) buffer at 4 °C for 30 min.

    Techniques: Chick Chorioallantoic Membrane Assay, Purification

    Proteolysis of goethite-associated BSA at 1.4 mg BSA m –2 , with or without coadsorbed phosphate, determined from either batch or IR experiments. The proteolysis was measured as the decrease of the intensity of the BSA peak in SEC ( cf. Figure 2 B,C) and the amide II band in the batch and IR experiment, respectively. All values were normalized against the initial value ( t = 0). The proteolysis of aqueous BSA at a concentration of 100 mg L –1 is shown for comparison. All experiments were performed at pH 4.0 in 0.01 M NaCl, and the protease concentration was 5 mg L –1 . Error bars represent standard deviations ( n = 3 for goethite-associated BSA and n = 2 for aqueous BSA).

    Journal: Environmental Science & Technology

    Article Title: Proteolysis of Iron Oxide-Associated Bovine Serum Albumin

    doi: 10.1021/acs.est.0c00860

    Figure Lengend Snippet: Proteolysis of goethite-associated BSA at 1.4 mg BSA m –2 , with or without coadsorbed phosphate, determined from either batch or IR experiments. The proteolysis was measured as the decrease of the intensity of the BSA peak in SEC ( cf. Figure 2 B,C) and the amide II band in the batch and IR experiment, respectively. All values were normalized against the initial value ( t = 0). The proteolysis of aqueous BSA at a concentration of 100 mg L –1 is shown for comparison. All experiments were performed at pH 4.0 in 0.01 M NaCl, and the protease concentration was 5 mg L –1 . Error bars represent standard deviations ( n = 3 for goethite-associated BSA and n = 2 for aqueous BSA).

    Article Snippet: After the reaction, the solid and the liquid phases were immediately separated by mixing 1 mL of iron oxide-BSA suspension with 0.2 mL of 5 M NaCl at pH 4.0 (to induce aggregation and facilitate solid–liquid separation) and centrifuged at 13,793g for 5 min (Heraeus Biofuge 13 centrifuge, Thermo Fisher Scientific, Waltham, MA).

    Techniques: Concentration Assay

    MCR analysis of IR spectral data sets of goethite-associated BSA during 20 h proteolysis reaction at 0.7 (low coverage, A) and 1.4 mg BSA m –2 (high coverage, B) and at pH 4.0 in 0.01 M NaCl. The spectra of components 1 (C1) and 2 (C2) are presented as dotted and solid lines, respectively, and the relative contribution of C1 and C2 during the proteolysis is shown as insets. Note that the spectra of C1 and C2 are presented as the unmodified output from the MCR analysis, that is, they have not been scaled separately after the analysis. The numbers in the plain and bold text indicate the main peak positions of C1 and C2, respectively. The first time point was collected after ca. 1 min.

    Journal: Environmental Science & Technology

    Article Title: Proteolysis of Iron Oxide-Associated Bovine Serum Albumin

    doi: 10.1021/acs.est.0c00860

    Figure Lengend Snippet: MCR analysis of IR spectral data sets of goethite-associated BSA during 20 h proteolysis reaction at 0.7 (low coverage, A) and 1.4 mg BSA m –2 (high coverage, B) and at pH 4.0 in 0.01 M NaCl. The spectra of components 1 (C1) and 2 (C2) are presented as dotted and solid lines, respectively, and the relative contribution of C1 and C2 during the proteolysis is shown as insets. Note that the spectra of C1 and C2 are presented as the unmodified output from the MCR analysis, that is, they have not been scaled separately after the analysis. The numbers in the plain and bold text indicate the main peak positions of C1 and C2, respectively. The first time point was collected after ca. 1 min.

    Article Snippet: After the reaction, the solid and the liquid phases were immediately separated by mixing 1 mL of iron oxide-BSA suspension with 0.2 mL of 5 M NaCl at pH 4.0 (to induce aggregation and facilitate solid–liquid separation) and centrifuged at 13,793g for 5 min (Heraeus Biofuge 13 centrifuge, Thermo Fisher Scientific, Waltham, MA).

    Techniques:

    Area-normalized SEC chromatograms of 100 mg L –1 BSA in aqueous solution (A) and the phosphate-desorbed fraction of ferrihydrite-associated BSA (B) and goethite-associated BSA (C), before ( T 0) and after 3 h proteolysis reaction ( T 3h). The experiments were performed at a BSA surface coverage of 1.4 mg m –2 (corresponding to total BSA concentrations of 200 and 100 mg L –1 in the ferrihydrite and goethite systems, respectively) at pH 4.0 in 0.01 M NaCl and at a protease concentration of 10 mg L –1 . The insets show the difference between the chromatograms at T 3h and T 0. The horizontal dotted lines in the insets indicate the 0-level. The shaded bands represent standard deviations ( n = 2). The molecular masses (in Da) of peptide standards are represented by the vertical dotted lines.

    Journal: Environmental Science & Technology

    Article Title: Proteolysis of Iron Oxide-Associated Bovine Serum Albumin

    doi: 10.1021/acs.est.0c00860

    Figure Lengend Snippet: Area-normalized SEC chromatograms of 100 mg L –1 BSA in aqueous solution (A) and the phosphate-desorbed fraction of ferrihydrite-associated BSA (B) and goethite-associated BSA (C), before ( T 0) and after 3 h proteolysis reaction ( T 3h). The experiments were performed at a BSA surface coverage of 1.4 mg m –2 (corresponding to total BSA concentrations of 200 and 100 mg L –1 in the ferrihydrite and goethite systems, respectively) at pH 4.0 in 0.01 M NaCl and at a protease concentration of 10 mg L –1 . The insets show the difference between the chromatograms at T 3h and T 0. The horizontal dotted lines in the insets indicate the 0-level. The shaded bands represent standard deviations ( n = 2). The molecular masses (in Da) of peptide standards are represented by the vertical dotted lines.

    Article Snippet: After the reaction, the solid and the liquid phases were immediately separated by mixing 1 mL of iron oxide-BSA suspension with 0.2 mL of 5 M NaCl at pH 4.0 (to induce aggregation and facilitate solid–liquid separation) and centrifuged at 13,793g for 5 min (Heraeus Biofuge 13 centrifuge, Thermo Fisher Scientific, Waltham, MA).

    Techniques: Concentration Assay

    Adsorption isotherms of BSA on ferrihydrite (A) and goethite (B). The experiments were conducted at pH 4.0 in 0.01 M NaCl for 24 h. The data are compared with a 1:1 line (dotted). Arrows indicate the total BSA concentrations added in the proteolysis experiments.

    Journal: Environmental Science & Technology

    Article Title: Proteolysis of Iron Oxide-Associated Bovine Serum Albumin

    doi: 10.1021/acs.est.0c00860

    Figure Lengend Snippet: Adsorption isotherms of BSA on ferrihydrite (A) and goethite (B). The experiments were conducted at pH 4.0 in 0.01 M NaCl for 24 h. The data are compared with a 1:1 line (dotted). Arrows indicate the total BSA concentrations added in the proteolysis experiments.

    Article Snippet: After the reaction, the solid and the liquid phases were immediately separated by mixing 1 mL of iron oxide-BSA suspension with 0.2 mL of 5 M NaCl at pH 4.0 (to induce aggregation and facilitate solid–liquid separation) and centrifuged at 13,793g for 5 min (Heraeus Biofuge 13 centrifuge, Thermo Fisher Scientific, Waltham, MA).

    Techniques: Adsorption

    Difference in SEC chromatograms of the phosphate-desorbed fraction of ferrihydrite-associated BSA (A) and goethite-associated BSA (B) in the presence of coadsorbed phosphate after 3 h proteolysis reaction. The experiments were performed at 0.7 mg BSA m –2 and pH 4.0 in 0.01 M NaCl, and the protease concentration was 10 mg L –1 . The total added phosphate concentrations were 1.0, 2.0, and 5.0 μmol m –2 , which are indicated by numbers in the figure legends. The difference chromatograms shown are obtained by subtraction with a control without phosphate addition. The horizontal dotted lines indicate the 0-level. The shaded bands represent standard deviations ( n = 2). The arrows indicate the direction of change, and the vertical dotted lines represent the molecular masses (in Da) of peptide standards.

    Journal: Environmental Science & Technology

    Article Title: Proteolysis of Iron Oxide-Associated Bovine Serum Albumin

    doi: 10.1021/acs.est.0c00860

    Figure Lengend Snippet: Difference in SEC chromatograms of the phosphate-desorbed fraction of ferrihydrite-associated BSA (A) and goethite-associated BSA (B) in the presence of coadsorbed phosphate after 3 h proteolysis reaction. The experiments were performed at 0.7 mg BSA m –2 and pH 4.0 in 0.01 M NaCl, and the protease concentration was 10 mg L –1 . The total added phosphate concentrations were 1.0, 2.0, and 5.0 μmol m –2 , which are indicated by numbers in the figure legends. The difference chromatograms shown are obtained by subtraction with a control without phosphate addition. The horizontal dotted lines indicate the 0-level. The shaded bands represent standard deviations ( n = 2). The arrows indicate the direction of change, and the vertical dotted lines represent the molecular masses (in Da) of peptide standards.

    Article Snippet: After the reaction, the solid and the liquid phases were immediately separated by mixing 1 mL of iron oxide-BSA suspension with 0.2 mL of 5 M NaCl at pH 4.0 (to induce aggregation and facilitate solid–liquid separation) and centrifuged at 13,793g for 5 min (Heraeus Biofuge 13 centrifuge, Thermo Fisher Scientific, Waltham, MA).

    Techniques: Concentration Assay

    CD spectra of the artificial WSTF PHD_EL5 RING finger and its five mutants. Spectra of 25 μM samples were collected in 20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, and 50 μM ZnCl 2 at room temperature. (1) K4R, (2) K8R, (3) K9R, (4) K14R, and (5) K23R are denoted by solid lines, and the dotted line displays the wild-type.

    Journal: Scientific Reports

    Article Title: Structural model of ubiquitin transfer onto an artificial RING finger as an E3 ligase

    doi: 10.1038/srep06574

    Figure Lengend Snippet: CD spectra of the artificial WSTF PHD_EL5 RING finger and its five mutants. Spectra of 25 μM samples were collected in 20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, and 50 μM ZnCl 2 at room temperature. (1) K4R, (2) K8R, (3) K9R, (4) K14R, and (5) K23R are denoted by solid lines, and the dotted line displays the wild-type.

    Article Snippet: The peptides dissolved in 1 ml of 8 M guanidine-HCl were dialyzed against degassed Solution A (20 mM Tris-HCl (pH 6.9), 50 mM NaCl, 1 mM dithiothreitol, 50 μM ZnCl2 ) overnight at 4°C using a Slide-A-Lyzer dialysis cassette (Thermo scientific, Rockford, IL, USA) as described previously .

    Techniques: