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  • 99
    Thermo Fisher 10x fastdigest buffer
    10x Fastdigest Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore 10x klentaq pcr buffer
    10x Klentaq Pcr Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche incubation buffer 10x
    Incubation Buffer 10x, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 10x fastap buffer
    Isolation and characterization of chaperone-bound luciferase from RL. Immunoisolation of chaperone complexes containing unfolded luciferase-myc-His (U) from control lysate and from lysate treated with HA or GA using chemically denatured ( A ) and thermally denatured ( B ) luciferase. In A , lanes 1 and 2, native luciferase (N) was added. In B , lanes 1 and 2, thermal denaturation of luciferase was carried out in ATP-depleted lysate. Complexes bound to protein G-Sepharose were eluted with ATP (A) or SDS (S). Eluted fractions were analyzed by SDS/PAGE followed by Coomassie blue staining ( Ai and B ) or immunoblotting with anti-Hsp90 and anti-Hsc70 ( Aii ). ( C ) Luciferase complexes were released from Sepharose beads with factor Xa and eluates immunoblotted with antibodies against Hip, Hsp40, and p23. ( D ) Time course of ATP-dependent elution of chaperones from complexes with luciferase isolated as in A from control lysate (−HA) and HA-treated lysate (+HA). Sepharose beads were incubated for 1 min at 25°C in 200 μl of <t>buffer</t> B with 1 mM ATP/5 mM Mg 2+ , and the supernatant was removed. This procedure was repeated five times, followed by a final elution with SDS. ATP eluates were analyzed as in A . Proteins were quantified by densitometry and plotted as the amount of total chaperone protein released up to a given time.
    10x Fastap Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 311 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa 10x h buffer
    Isolation and characterization of chaperone-bound luciferase from RL. Immunoisolation of chaperone complexes containing unfolded luciferase-myc-His (U) from control lysate and from lysate treated with HA or GA using chemically denatured ( A ) and thermally denatured ( B ) luciferase. In A , lanes 1 and 2, native luciferase (N) was added. In B , lanes 1 and 2, thermal denaturation of luciferase was carried out in ATP-depleted lysate. Complexes bound to protein G-Sepharose were eluted with ATP (A) or SDS (S). Eluted fractions were analyzed by SDS/PAGE followed by Coomassie blue staining ( Ai and B ) or immunoblotting with anti-Hsp90 and anti-Hsc70 ( Aii ). ( C ) Luciferase complexes were released from Sepharose beads with factor Xa and eluates immunoblotted with antibodies against Hip, Hsp40, and p23. ( D ) Time course of ATP-dependent elution of chaperones from complexes with luciferase isolated as in A from control lysate (−HA) and HA-treated lysate (+HA). Sepharose beads were incubated for 1 min at 25°C in 200 μl of <t>buffer</t> B with 1 mM ATP/5 mM Mg 2+ , and the supernatant was removed. This procedure was repeated five times, followed by a final elution with SDS. ATP eluates were analyzed as in A . Proteins were quantified by densitometry and plotted as the amount of total chaperone protein released up to a given time.
    10x H Buffer, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam buffer
    Isolation and characterization of chaperone-bound luciferase from RL. Immunoisolation of chaperone complexes containing unfolded luciferase-myc-His (U) from control lysate and from lysate treated with HA or GA using chemically denatured ( A ) and thermally denatured ( B ) luciferase. In A , lanes 1 and 2, native luciferase (N) was added. In B , lanes 1 and 2, thermal denaturation of luciferase was carried out in ATP-depleted lysate. Complexes bound to protein G-Sepharose were eluted with ATP (A) or SDS (S). Eluted fractions were analyzed by SDS/PAGE followed by Coomassie blue staining ( Ai and B ) or immunoblotting with anti-Hsp90 and anti-Hsc70 ( Aii ). ( C ) Luciferase complexes were released from Sepharose beads with factor Xa and eluates immunoblotted with antibodies against Hip, Hsp40, and p23. ( D ) Time course of ATP-dependent elution of chaperones from complexes with luciferase isolated as in A from control lysate (−HA) and HA-treated lysate (+HA). Sepharose beads were incubated for 1 min at 25°C in 200 μl of <t>buffer</t> B with 1 mM ATP/5 mM Mg 2+ , and the supernatant was removed. This procedure was repeated five times, followed by a final elution with SDS. ATP eluates were analyzed as in A . Proteins were quantified by densitometry and plotted as the amount of total chaperone protein released up to a given time.
    Buffer, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2865 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc ripa buffer
    Insulin-induced interaction between IRK and Neu1 on the cell surface. A : Colocalization of IRK and Neu1 on the plasma membrane. HEK cells expressing Neu1-YFP and IRK-hRluc chimeric proteins were fixed with paraformaldehyde, permeabilized by Triton X-100, and costained with mouse anti-insulin receptor β-chain (Cell Signaling) (1:400) and rabbit anti-Neu1 (1:100) antibodies. Cells were then counterstained with Oregon Green 488–conjugated anti-rabbit IgG antibodies and Texas Red–conjugated goat anti-mouse antibodies (1:1,000; Molecular Probes). Slides were studied on a Zeiss LSM510 inverted confocal microscope. B : Detection of interaction between IRK and Neu1 by BRET. HEK cells expressing Neu1-YFP and IRK-hRluc ( left ) or YFP and IRK-hRluc ( right ) were treated with coelenterazine H in the absence or in the presence of 5 and 50 nmol/L insulin, and the BRET signal was calculated as the ratio of light intensity emitted by IRK-hRluc (530 nm) and Neu1-YFP (480 nm). In the absence of insulin, only the background levels of energy transfer between Neu1-YFP and IRK-hRluc are observed, whereas incubation of cells with insulin at physiological concentration of 5 nmol/L results in the appearance of BRET signal. C : Coimmunoprecipitation of IRK and Neu1. Hek293T cells overexpressing IRK-hRluc, Neu1-YFP, and CathA plasmids were treated or not with 20 nmol/L insulin for 15 min, washed with ice-cold <t>PBS,</t> and lysed in <t>RIPA</t> buffer containing dithiobis(succinimidyl propionate) cross-linker. After removal of cell debris by centrifugation, supernatants were subjected to immunoprecipitation using anti-insulin receptor β-chain antibodies. Proteins in cell lysate (L), supernatant of cell extract (S), protein A agarose washes (W), and eluate (E) were analyzed by Western blotting using anti-insulin receptor β-chain and anti-Neu1 antibodies.
    Ripa Buffer, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 6367 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher buffer tango
    Insulin-induced interaction between IRK and Neu1 on the cell surface. A : Colocalization of IRK and Neu1 on the plasma membrane. HEK cells expressing Neu1-YFP and IRK-hRluc chimeric proteins were fixed with paraformaldehyde, permeabilized by Triton X-100, and costained with mouse anti-insulin receptor β-chain (Cell Signaling) (1:400) and rabbit anti-Neu1 (1:100) antibodies. Cells were then counterstained with Oregon Green 488–conjugated anti-rabbit IgG antibodies and Texas Red–conjugated goat anti-mouse antibodies (1:1,000; Molecular Probes). Slides were studied on a Zeiss LSM510 inverted confocal microscope. B : Detection of interaction between IRK and Neu1 by BRET. HEK cells expressing Neu1-YFP and IRK-hRluc ( left ) or YFP and IRK-hRluc ( right ) were treated with coelenterazine H in the absence or in the presence of 5 and 50 nmol/L insulin, and the BRET signal was calculated as the ratio of light intensity emitted by IRK-hRluc (530 nm) and Neu1-YFP (480 nm). In the absence of insulin, only the background levels of energy transfer between Neu1-YFP and IRK-hRluc are observed, whereas incubation of cells with insulin at physiological concentration of 5 nmol/L results in the appearance of BRET signal. C : Coimmunoprecipitation of IRK and Neu1. Hek293T cells overexpressing IRK-hRluc, Neu1-YFP, and CathA plasmids were treated or not with 20 nmol/L insulin for 15 min, washed with ice-cold <t>PBS,</t> and lysed in <t>RIPA</t> buffer containing dithiobis(succinimidyl propionate) cross-linker. After removal of cell debris by centrifugation, supernatants were subjected to immunoprecipitation using anti-insulin receptor β-chain antibodies. Proteins in cell lysate (L), supernatant of cell extract (S), protein A agarose washes (W), and eluate (E) were analyzed by Western blotting using anti-insulin receptor β-chain and anti-Neu1 antibodies.
    Buffer Tango, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher r buffer a
    Insulin-induced interaction between IRK and Neu1 on the cell surface. A : Colocalization of IRK and Neu1 on the plasma membrane. HEK cells expressing Neu1-YFP and IRK-hRluc chimeric proteins were fixed with paraformaldehyde, permeabilized by Triton X-100, and costained with mouse anti-insulin receptor β-chain (Cell Signaling) (1:400) and rabbit anti-Neu1 (1:100) antibodies. Cells were then counterstained with Oregon Green 488–conjugated anti-rabbit IgG antibodies and Texas Red–conjugated goat anti-mouse antibodies (1:1,000; Molecular Probes). Slides were studied on a Zeiss LSM510 inverted confocal microscope. B : Detection of interaction between IRK and Neu1 by BRET. HEK cells expressing Neu1-YFP and IRK-hRluc ( left ) or YFP and IRK-hRluc ( right ) were treated with coelenterazine H in the absence or in the presence of 5 and 50 nmol/L insulin, and the BRET signal was calculated as the ratio of light intensity emitted by IRK-hRluc (530 nm) and Neu1-YFP (480 nm). In the absence of insulin, only the background levels of energy transfer between Neu1-YFP and IRK-hRluc are observed, whereas incubation of cells with insulin at physiological concentration of 5 nmol/L results in the appearance of BRET signal. C : Coimmunoprecipitation of IRK and Neu1. Hek293T cells overexpressing IRK-hRluc, Neu1-YFP, and CathA plasmids were treated or not with 20 nmol/L insulin for 15 min, washed with ice-cold <t>PBS,</t> and lysed in <t>RIPA</t> buffer containing dithiobis(succinimidyl propionate) cross-linker. After removal of cell debris by centrifugation, supernatants were subjected to immunoprecipitation using anti-insulin receptor β-chain antibodies. Proteins in cell lysate (L), supernatant of cell extract (S), protein A agarose washes (W), and eluate (E) were analyzed by Western blotting using anti-insulin receptor β-chain and anti-Neu1 antibodies.
    R Buffer A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher buffer o
    Insulin-induced interaction between IRK and Neu1 on the cell surface. A : Colocalization of IRK and Neu1 on the plasma membrane. HEK cells expressing Neu1-YFP and IRK-hRluc chimeric proteins were fixed with paraformaldehyde, permeabilized by Triton X-100, and costained with mouse anti-insulin receptor β-chain (Cell Signaling) (1:400) and rabbit anti-Neu1 (1:100) antibodies. Cells were then counterstained with Oregon Green 488–conjugated anti-rabbit IgG antibodies and Texas Red–conjugated goat anti-mouse antibodies (1:1,000; Molecular Probes). Slides were studied on a Zeiss LSM510 inverted confocal microscope. B : Detection of interaction between IRK and Neu1 by BRET. HEK cells expressing Neu1-YFP and IRK-hRluc ( left ) or YFP and IRK-hRluc ( right ) were treated with coelenterazine H in the absence or in the presence of 5 and 50 nmol/L insulin, and the BRET signal was calculated as the ratio of light intensity emitted by IRK-hRluc (530 nm) and Neu1-YFP (480 nm). In the absence of insulin, only the background levels of energy transfer between Neu1-YFP and IRK-hRluc are observed, whereas incubation of cells with insulin at physiological concentration of 5 nmol/L results in the appearance of BRET signal. C : Coimmunoprecipitation of IRK and Neu1. Hek293T cells overexpressing IRK-hRluc, Neu1-YFP, and CathA plasmids were treated or not with 20 nmol/L insulin for 15 min, washed with ice-cold <t>PBS,</t> and lysed in <t>RIPA</t> buffer containing dithiobis(succinimidyl propionate) cross-linker. After removal of cell debris by centrifugation, supernatants were subjected to immunoprecipitation using anti-insulin receptor β-chain antibodies. Proteins in cell lysate (L), supernatant of cell extract (S), protein A agarose washes (W), and eluate (E) were analyzed by Western blotting using anti-insulin receptor β-chain and anti-Neu1 antibodies.
    Buffer O, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqi buffer
    Insulin-induced interaction between IRK and Neu1 on the cell surface. A : Colocalization of IRK and Neu1 on the plasma membrane. HEK cells expressing Neu1-YFP and IRK-hRluc chimeric proteins were fixed with paraformaldehyde, permeabilized by Triton X-100, and costained with mouse anti-insulin receptor β-chain (Cell Signaling) (1:400) and rabbit anti-Neu1 (1:100) antibodies. Cells were then counterstained with Oregon Green 488–conjugated anti-rabbit IgG antibodies and Texas Red–conjugated goat anti-mouse antibodies (1:1,000; Molecular Probes). Slides were studied on a Zeiss LSM510 inverted confocal microscope. B : Detection of interaction between IRK and Neu1 by BRET. HEK cells expressing Neu1-YFP and IRK-hRluc ( left ) or YFP and IRK-hRluc ( right ) were treated with coelenterazine H in the absence or in the presence of 5 and 50 nmol/L insulin, and the BRET signal was calculated as the ratio of light intensity emitted by IRK-hRluc (530 nm) and Neu1-YFP (480 nm). In the absence of insulin, only the background levels of energy transfer between Neu1-YFP and IRK-hRluc are observed, whereas incubation of cells with insulin at physiological concentration of 5 nmol/L results in the appearance of BRET signal. C : Coimmunoprecipitation of IRK and Neu1. Hek293T cells overexpressing IRK-hRluc, Neu1-YFP, and CathA plasmids were treated or not with 20 nmol/L insulin for 15 min, washed with ice-cold <t>PBS,</t> and lysed in <t>RIPA</t> buffer containing dithiobis(succinimidyl propionate) cross-linker. After removal of cell debris by centrifugation, supernatants were subjected to immunoprecipitation using anti-insulin receptor β-chain antibodies. Proteins in cell lysate (L), supernatant of cell extract (S), protein A agarose washes (W), and eluate (E) were analyzed by Western blotting using anti-insulin receptor β-chain and anti-Neu1 antibodies.
    Taqi Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher buffer g
    Insulin-induced interaction between IRK and Neu1 on the cell surface. A : Colocalization of IRK and Neu1 on the plasma membrane. HEK cells expressing Neu1-YFP and IRK-hRluc chimeric proteins were fixed with paraformaldehyde, permeabilized by Triton X-100, and costained with mouse anti-insulin receptor β-chain (Cell Signaling) (1:400) and rabbit anti-Neu1 (1:100) antibodies. Cells were then counterstained with Oregon Green 488–conjugated anti-rabbit IgG antibodies and Texas Red–conjugated goat anti-mouse antibodies (1:1,000; Molecular Probes). Slides were studied on a Zeiss LSM510 inverted confocal microscope. B : Detection of interaction between IRK and Neu1 by BRET. HEK cells expressing Neu1-YFP and IRK-hRluc ( left ) or YFP and IRK-hRluc ( right ) were treated with coelenterazine H in the absence or in the presence of 5 and 50 nmol/L insulin, and the BRET signal was calculated as the ratio of light intensity emitted by IRK-hRluc (530 nm) and Neu1-YFP (480 nm). In the absence of insulin, only the background levels of energy transfer between Neu1-YFP and IRK-hRluc are observed, whereas incubation of cells with insulin at physiological concentration of 5 nmol/L results in the appearance of BRET signal. C : Coimmunoprecipitation of IRK and Neu1. Hek293T cells overexpressing IRK-hRluc, Neu1-YFP, and CathA plasmids were treated or not with 20 nmol/L insulin for 15 min, washed with ice-cold <t>PBS,</t> and lysed in <t>RIPA</t> buffer containing dithiobis(succinimidyl propionate) cross-linker. After removal of cell debris by centrifugation, supernatants were subjected to immunoprecipitation using anti-insulin receptor β-chain antibodies. Proteins in cell lysate (L), supernatant of cell extract (S), protein A agarose washes (W), and eluate (E) were analyzed by Western blotting using anti-insulin receptor β-chain and anti-Neu1 antibodies.
    Buffer G, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dreamtaq buffer
    Insulin-induced interaction between IRK and Neu1 on the cell surface. A : Colocalization of IRK and Neu1 on the plasma membrane. HEK cells expressing Neu1-YFP and IRK-hRluc chimeric proteins were fixed with paraformaldehyde, permeabilized by Triton X-100, and costained with mouse anti-insulin receptor β-chain (Cell Signaling) (1:400) and rabbit anti-Neu1 (1:100) antibodies. Cells were then counterstained with Oregon Green 488–conjugated anti-rabbit IgG antibodies and Texas Red–conjugated goat anti-mouse antibodies (1:1,000; Molecular Probes). Slides were studied on a Zeiss LSM510 inverted confocal microscope. B : Detection of interaction between IRK and Neu1 by BRET. HEK cells expressing Neu1-YFP and IRK-hRluc ( left ) or YFP and IRK-hRluc ( right ) were treated with coelenterazine H in the absence or in the presence of 5 and 50 nmol/L insulin, and the BRET signal was calculated as the ratio of light intensity emitted by IRK-hRluc (530 nm) and Neu1-YFP (480 nm). In the absence of insulin, only the background levels of energy transfer between Neu1-YFP and IRK-hRluc are observed, whereas incubation of cells with insulin at physiological concentration of 5 nmol/L results in the appearance of BRET signal. C : Coimmunoprecipitation of IRK and Neu1. Hek293T cells overexpressing IRK-hRluc, Neu1-YFP, and CathA plasmids were treated or not with 20 nmol/L insulin for 15 min, washed with ice-cold <t>PBS,</t> and lysed in <t>RIPA</t> buffer containing dithiobis(succinimidyl propionate) cross-linker. After removal of cell debris by centrifugation, supernatants were subjected to immunoprecipitation using anti-insulin receptor β-chain antibodies. Proteins in cell lysate (L), supernatant of cell extract (S), protein A agarose washes (W), and eluate (E) were analyzed by Western blotting using anti-insulin receptor β-chain and anti-Neu1 antibodies.
    Dreamtaq Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher buffer ecori
    Confirmation of glpD disruption. A ) Southern blot analysis confirms the disruption of glpD . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with <t>BamHI</t> and blotted on a nylon membrane. Digoxygenein-11-dUTP labeled bb0243 probe was utilized to visualize glpD . Migration positions of DNA molecular size markers are indicated on the left. B ) glpD disruption occurred via by a double crossover insertion of flgB - aadA . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI or <t>EcoRI,</t> which would be specific for the proximal or distal bb0243 flanking chromosomal regions, respectively, and blotted to a nylon membrane. Blots were developed with a digoxygenein-11-dUTP-labeled aadA probe. A schematic diagram indicates the sizes of the fragments expected to contain flgB - aadA . Migration positions of DNA molecular size markers are indicated on the left of each panel.
    Buffer Ecori, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 900 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad buffer
    Confirmation of glpD disruption. A ) Southern blot analysis confirms the disruption of glpD . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with <t>BamHI</t> and blotted on a nylon membrane. Digoxygenein-11-dUTP labeled bb0243 probe was utilized to visualize glpD . Migration positions of DNA molecular size markers are indicated on the left. B ) glpD disruption occurred via by a double crossover insertion of flgB - aadA . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI or <t>EcoRI,</t> which would be specific for the proximal or distal bb0243 flanking chromosomal regions, respectively, and blotted to a nylon membrane. Blots were developed with a digoxygenein-11-dUTP-labeled aadA probe. A schematic diagram indicates the sizes of the fragments expected to contain flgB - aadA . Migration positions of DNA molecular size markers are indicated on the left of each panel.
    Buffer, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 11897 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 10x buffer
    Confirmation of glpD disruption. A ) Southern blot analysis confirms the disruption of glpD . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with <t>BamHI</t> and blotted on a nylon membrane. Digoxygenein-11-dUTP labeled bb0243 probe was utilized to visualize glpD . Migration positions of DNA molecular size markers are indicated on the left. B ) glpD disruption occurred via by a double crossover insertion of flgB - aadA . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI or <t>EcoRI,</t> which would be specific for the proximal or distal bb0243 flanking chromosomal regions, respectively, and blotted to a nylon membrane. Blots were developed with a digoxygenein-11-dUTP-labeled aadA probe. A schematic diagram indicates the sizes of the fragments expected to contain flgB - aadA . Migration positions of DNA molecular size markers are indicated on the left of each panel.
    10x Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 681 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hybridization buffer
    Confirmation of glpD disruption. A ) Southern blot analysis confirms the disruption of glpD . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with <t>BamHI</t> and blotted on a nylon membrane. Digoxygenein-11-dUTP labeled bb0243 probe was utilized to visualize glpD . Migration positions of DNA molecular size markers are indicated on the left. B ) glpD disruption occurred via by a double crossover insertion of flgB - aadA . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI or <t>EcoRI,</t> which would be specific for the proximal or distal bb0243 flanking chromosomal regions, respectively, and blotted to a nylon membrane. Blots were developed with a digoxygenein-11-dUTP-labeled aadA probe. A schematic diagram indicates the sizes of the fragments expected to contain flgB - aadA . Migration positions of DNA molecular size markers are indicated on the left of each panel.
    Hybridization Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 3003 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PRDM9, EWSR1, and CDYL coimmunoprecipitate each other and chromosomal axis/synaptonemal complex proteins from wild-type 14-dpp spermatocytes. (A) PRDM9 and its interactors coimmunoprecipitate each other. Left, <t>coIP</t> with anti-PRDM9; middle, coIP with anti-EWSR1; right, coIP with CDYL. Lane 1, input, 10 µg (2.5 µg in the CDYL and EHMT2 blots); lane 2, IgG, 10 µg, coIP with nonimmune IgG; lanes 2 and 3, αPRDM9, αEWSR1, and αCDYL, 10 µg, coIP with the respective antibody, either nontreated (lane 3) or <t>DNase</t> treated (lane 4). The antibodies used to detect specific proteins on Western blots are shown on the left. (B) PRDM9 and its interactors also interact with chromosomal axis/SC proteins. coIP with the same antibodies used in A but probed with antibodies against chromosome axis/SC proteins as marked on the left. (C) Reciprocal coIP shows that SC proteins coimmunoprecipitate PRDM9 and its interactors. coIP with SYCP3 (left) or SYCP1 (right). The antibodies used for detection on Western blots are on the left. (D) Interaction between REC8 and PRDM9 in cultured cells. REC8 was cloned under a V5 tag, and PRDM9 was cloned under a FLAG tag. The two proteins were either coexpressed or separately expressed in HEK 293 cells. Specific interaction between the two proteins was detected in extracts of cells in which the two proteins were coexpressed (Co, left) but not in mixed extracts of cells in which the two proteins were expressed separately (Mix, right). Note that the interacting band is the middle of three bands, indicating that it is a phosphorylated form.
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    PRDM9, EWSR1, and CDYL coimmunoprecipitate each other and chromosomal axis/synaptonemal complex proteins from wild-type 14-dpp spermatocytes. (A) PRDM9 and its interactors coimmunoprecipitate each other. Left, <t>coIP</t> with anti-PRDM9; middle, coIP with anti-EWSR1; right, coIP with CDYL. Lane 1, input, 10 µg (2.5 µg in the CDYL and EHMT2 blots); lane 2, IgG, 10 µg, coIP with nonimmune IgG; lanes 2 and 3, αPRDM9, αEWSR1, and αCDYL, 10 µg, coIP with the respective antibody, either nontreated (lane 3) or <t>DNase</t> treated (lane 4). The antibodies used to detect specific proteins on Western blots are shown on the left. (B) PRDM9 and its interactors also interact with chromosomal axis/SC proteins. coIP with the same antibodies used in A but probed with antibodies against chromosome axis/SC proteins as marked on the left. (C) Reciprocal coIP shows that SC proteins coimmunoprecipitate PRDM9 and its interactors. coIP with SYCP3 (left) or SYCP1 (right). The antibodies used for detection on Western blots are on the left. (D) Interaction between REC8 and PRDM9 in cultured cells. REC8 was cloned under a V5 tag, and PRDM9 was cloned under a FLAG tag. The two proteins were either coexpressed or separately expressed in HEK 293 cells. Specific interaction between the two proteins was detected in extracts of cells in which the two proteins were coexpressed (Co, left) but not in mixed extracts of cells in which the two proteins were expressed separately (Mix, right). Note that the interacting band is the middle of three bands, indicating that it is a phosphorylated form.
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    Cellular function reinstitution of genome-edited <t>RBCs.</t> a For cellular function study, in vitro <t>RBC</t> differentiation of cloned erythroid progenitor cells was induced. Nuclei of the resultant cells were initially stained with Hoechst dye and examined under microscope, demonstrating induced in vitro RBC differentiation and maturation. b Cytospin of resultant cells with Wright-Giemsa staining confirmed presence of mature RBCs (no nuclei), RBC with nucleus contraction, and nucleated RBC. c Cell sickling assays of in vitro differentiated RBCs with or without genome editing, and RBCs from the SCD, SCT, and healthy donor blood demonstrated that CRISPR genome editing of patient HSPCs resulted in function reinstitution of the cloned offspring RBCs, which became more resistant to hypoxia. d Time course of sickling cell formation (%). The data are from the average of three clones
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    Cellular function reinstitution of genome-edited <t>RBCs.</t> a For cellular function study, in vitro <t>RBC</t> differentiation of cloned erythroid progenitor cells was induced. Nuclei of the resultant cells were initially stained with Hoechst dye and examined under microscope, demonstrating induced in vitro RBC differentiation and maturation. b Cytospin of resultant cells with Wright-Giemsa staining confirmed presence of mature RBCs (no nuclei), RBC with nucleus contraction, and nucleated RBC. c Cell sickling assays of in vitro differentiated RBCs with or without genome editing, and RBCs from the SCD, SCT, and healthy donor blood demonstrated that CRISPR genome editing of patient HSPCs resulted in function reinstitution of the cloned offspring RBCs, which became more resistant to hypoxia. d Time course of sickling cell formation (%). The data are from the average of three clones
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    Cellular function reinstitution of genome-edited <t>RBCs.</t> a For cellular function study, in vitro <t>RBC</t> differentiation of cloned erythroid progenitor cells was induced. Nuclei of the resultant cells were initially stained with Hoechst dye and examined under microscope, demonstrating induced in vitro RBC differentiation and maturation. b Cytospin of resultant cells with Wright-Giemsa staining confirmed presence of mature RBCs (no nuclei), RBC with nucleus contraction, and nucleated RBC. c Cell sickling assays of in vitro differentiated RBCs with or without genome editing, and RBCs from the SCD, SCT, and healthy donor blood demonstrated that CRISPR genome editing of patient HSPCs resulted in function reinstitution of the cloned offspring RBCs, which became more resistant to hypoxia. d Time course of sickling cell formation (%). The data are from the average of three clones
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    Cellular function reinstitution of genome-edited <t>RBCs.</t> a For cellular function study, in vitro <t>RBC</t> differentiation of cloned erythroid progenitor cells was induced. Nuclei of the resultant cells were initially stained with Hoechst dye and examined under microscope, demonstrating induced in vitro RBC differentiation and maturation. b Cytospin of resultant cells with Wright-Giemsa staining confirmed presence of mature RBCs (no nuclei), RBC with nucleus contraction, and nucleated RBC. c Cell sickling assays of in vitro differentiated RBCs with or without genome editing, and RBCs from the SCD, SCT, and healthy donor blood demonstrated that CRISPR genome editing of patient HSPCs resulted in function reinstitution of the cloned offspring RBCs, which became more resistant to hypoxia. d Time course of sickling cell formation (%). The data are from the average of three clones
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    Trib1 overexpression in B cells induces a reduction in the production of secreted form of IgG1. Total splenocytes from Trib1-ROSA and Trib1-ROSA Mb1Cre mice were stimulated with LPS/IL-4 for 72 h in vitro [with (+) or without (Ø) the addition of a protein transport inhibitor “GolgiStop™” for the last 8 h of culture], then stained for intracytoplasmic IgG1 after a step of membrane Ig blocking using an anti-murine IgG antibody, a step of fixation and <t>permeabilization,</t> and finally were analyzed by flow cytometry. (A) Percentage of B220 + B cells stained for intracellular IgG1. (B) MFI of intracellular IgG1 staining on B220 + B cells. (C) A representative sample of each condition is shown. Each dot represents the result for one animal.
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    Trib1 overexpression in B cells induces a reduction in the production of secreted form of IgG1. Total splenocytes from Trib1-ROSA and Trib1-ROSA Mb1Cre mice were stimulated with LPS/IL-4 for 72 h in vitro [with (+) or without (Ø) the addition of a protein transport inhibitor “GolgiStop™” for the last 8 h of culture], then stained for intracytoplasmic IgG1 after a step of membrane Ig blocking using an anti-murine IgG antibody, a step of fixation and <t>permeabilization,</t> and finally were analyzed by flow cytometry. (A) Percentage of B220 + B cells stained for intracellular IgG1. (B) MFI of intracellular IgG1 staining on B220 + B cells. (C) A representative sample of each condition is shown. Each dot represents the result for one animal.
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    T regulatory cells are increased in infants On d14 p.i., the tracheobronchial lymph nodes (TBLN) (A), lung (B) and spleen (C) were isolated and processed to yield single cell suspensions. S pleens were also isolated from non-infected animals (C) . Cells were stained for CD4 + and <t>FoxP3</t> + . Averaged data shown are the percentage of FoxP3-expressing cells within the CD4 + population. Significance at each timepoint was determined using a student’s t test. *p≤0.05, **p ≤ 0.01.
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    Image Search Results


    Isolation and characterization of chaperone-bound luciferase from RL. Immunoisolation of chaperone complexes containing unfolded luciferase-myc-His (U) from control lysate and from lysate treated with HA or GA using chemically denatured ( A ) and thermally denatured ( B ) luciferase. In A , lanes 1 and 2, native luciferase (N) was added. In B , lanes 1 and 2, thermal denaturation of luciferase was carried out in ATP-depleted lysate. Complexes bound to protein G-Sepharose were eluted with ATP (A) or SDS (S). Eluted fractions were analyzed by SDS/PAGE followed by Coomassie blue staining ( Ai and B ) or immunoblotting with anti-Hsp90 and anti-Hsc70 ( Aii ). ( C ) Luciferase complexes were released from Sepharose beads with factor Xa and eluates immunoblotted with antibodies against Hip, Hsp40, and p23. ( D ) Time course of ATP-dependent elution of chaperones from complexes with luciferase isolated as in A from control lysate (−HA) and HA-treated lysate (+HA). Sepharose beads were incubated for 1 min at 25°C in 200 μl of buffer B with 1 mM ATP/5 mM Mg 2+ , and the supernatant was removed. This procedure was repeated five times, followed by a final elution with SDS. ATP eluates were analyzed as in A . Proteins were quantified by densitometry and plotted as the amount of total chaperone protein released up to a given time.

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

    Article Title: Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90

    doi:

    Figure Lengend Snippet: Isolation and characterization of chaperone-bound luciferase from RL. Immunoisolation of chaperone complexes containing unfolded luciferase-myc-His (U) from control lysate and from lysate treated with HA or GA using chemically denatured ( A ) and thermally denatured ( B ) luciferase. In A , lanes 1 and 2, native luciferase (N) was added. In B , lanes 1 and 2, thermal denaturation of luciferase was carried out in ATP-depleted lysate. Complexes bound to protein G-Sepharose were eluted with ATP (A) or SDS (S). Eluted fractions were analyzed by SDS/PAGE followed by Coomassie blue staining ( Ai and B ) or immunoblotting with anti-Hsp90 and anti-Hsc70 ( Aii ). ( C ) Luciferase complexes were released from Sepharose beads with factor Xa and eluates immunoblotted with antibodies against Hip, Hsp40, and p23. ( D ) Time course of ATP-dependent elution of chaperones from complexes with luciferase isolated as in A from control lysate (−HA) and HA-treated lysate (+HA). Sepharose beads were incubated for 1 min at 25°C in 200 μl of buffer B with 1 mM ATP/5 mM Mg 2+ , and the supernatant was removed. This procedure was repeated five times, followed by a final elution with SDS. ATP eluates were analyzed as in A . Proteins were quantified by densitometry and plotted as the amount of total chaperone protein released up to a given time.

    Article Snippet: RL (Green Hectares, Oregon, WI) was desalted on a 9.1-ml G25-Sephadex column (Pharmacia) into buffer B (20 mM Hepes·KOH, pH 7.4/100 mM potassium acetate/5% glycerol) and incubated for 30 min at 25°C either with 0.2% DMSO (control), 36 μM HA, or 36 μM GA (GIBCO) (added from a 17-mM solution in DMSO), or with the ansamycin concentrations indicated (0.2% DMSO had no effect on refolding).

    Techniques: Isolation, Luciferase, SDS Page, Staining, Incubation

    Induction of TKT transcription by Zta is revealed by RT-PCR and Northern blotting. (A) Detection of TKT transcripts in Zta-expressing transient transfectants or in stable clones of NPC-TW01 and RHEK-1 cells using RT-PCR. The reaction without reverse transcriptase served as a control to rule out the possibility of contamination with genomic DNA. The reaction detecting hypoxanthine phosphoribosyltransferase (HP) transcripts was an internal control for RNA quality and amounts. (B) Detection of TKT transcripts by RT-PCR in HONE-1-Zta cells, with or without induction by 1 μg of doxycycline per ml, using the same procedure. (C) Northern blot analysis of TKT expression in cells expressing Zta. mRNA (2 μg) from Zta-expressing clones NPC-TW01-Z, RHEK-Z2, and RHEK-Z3 and vector controls was subjected to agarose gel electrophoresis (0.8% agarose), blotted, and hybridized with a 32 P-labeled TKT probe as described in Materials and Methods. Human placental mRNA (lane P) served as a positive control. The lane marked with an asterisk was exposed for a shorter period. The same blot was probed with GAPDH as an internal control. The numbers to the left indicate the molecular size markers in kilobases.

    Journal: Journal of Virology

    Article Title: Upregulation of Tyrosine Kinase TKT by the Epstein-Barr Virus Transactivator Zta

    doi:

    Figure Lengend Snippet: Induction of TKT transcription by Zta is revealed by RT-PCR and Northern blotting. (A) Detection of TKT transcripts in Zta-expressing transient transfectants or in stable clones of NPC-TW01 and RHEK-1 cells using RT-PCR. The reaction without reverse transcriptase served as a control to rule out the possibility of contamination with genomic DNA. The reaction detecting hypoxanthine phosphoribosyltransferase (HP) transcripts was an internal control for RNA quality and amounts. (B) Detection of TKT transcripts by RT-PCR in HONE-1-Zta cells, with or without induction by 1 μg of doxycycline per ml, using the same procedure. (C) Northern blot analysis of TKT expression in cells expressing Zta. mRNA (2 μg) from Zta-expressing clones NPC-TW01-Z, RHEK-Z2, and RHEK-Z3 and vector controls was subjected to agarose gel electrophoresis (0.8% agarose), blotted, and hybridized with a 32 P-labeled TKT probe as described in Materials and Methods. Human placental mRNA (lane P) served as a positive control. The lane marked with an asterisk was exposed for a shorter period. The same blot was probed with GAPDH as an internal control. The numbers to the left indicate the molecular size markers in kilobases.

    Article Snippet: For qualitative PCR amplification, 25-μl reaction mixtures containing 2.5 μl of cDNA, 0.2 mM dNTP, 0.2 μM primers, 1× PCR buffer, and 1 U of Dynazyme II DNA polymerase (Finnzymes, Oy, Finland) were amplified for 25 or 40 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Northern Blot, Expressing, Clone Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Labeling, Positive Control

    Expression and status of TKT protein. (A) Detection of TKT protein in Zta-expressing cells by Western blot analysis. Total protein (20 μg) was extracted from Zta-expressing transfectants RHEK-Z2, RHEK-Z3, and NPC-TW01-Z and control cells and was blotted and reacted with a TKT-specific antibody. After being incubated with peroxidase-labeled goat anti-mouse immunoglobulin G, the membrane was visualized with a chemiluminescence kit. The same blot was reacted with Zta-specific antibody 4F10 to measure Zta expression and with the tubulin-specific antibody as an internal control. The two lanes marked with asterisks contained NPC-TW01 cells transfected with vector control (lane V∗) or transfected with pRc/CMV-TKT plasmid (lane TKT∗) and were the controls for TKT expression. (B) Phosphorylation status of TKT in Zta-expressing stable clones. RHEK vector control (lane V) and RHEK-Z2 (lane Z2) cells were immunoprecipitated with antiphosphotyrosine antibody (4G10) or irrelevant antibody control (con), subjected to SDS-PAGE (10% polyacrylamide), and blotted with anti-TKT antibody as described in Materials and Methods. The numbers to the left indicate the molecular mass markers in kilodaltons. (C) Expression of the TKT downstream effector MMP-1 transcripts detected by RT-PCR. RNA extracted from RHEK parental (lane P), vector control (lane V), and RHEK-Z2 (lane Z2) cells was subjected to RT and amplified by PCR using Zta-, TKT-, MMP-1-, and hypoxanthine phosphoribosyltransferase (HP)-specific primers. The reaction without reverse transcriptase served as a control to rule out the possibility of contamination with genomic DNA. The reaction detecting HP transcripts was an internal control for RNA quality and amounts.

    Journal: Journal of Virology

    Article Title: Upregulation of Tyrosine Kinase TKT by the Epstein-Barr Virus Transactivator Zta

    doi:

    Figure Lengend Snippet: Expression and status of TKT protein. (A) Detection of TKT protein in Zta-expressing cells by Western blot analysis. Total protein (20 μg) was extracted from Zta-expressing transfectants RHEK-Z2, RHEK-Z3, and NPC-TW01-Z and control cells and was blotted and reacted with a TKT-specific antibody. After being incubated with peroxidase-labeled goat anti-mouse immunoglobulin G, the membrane was visualized with a chemiluminescence kit. The same blot was reacted with Zta-specific antibody 4F10 to measure Zta expression and with the tubulin-specific antibody as an internal control. The two lanes marked with asterisks contained NPC-TW01 cells transfected with vector control (lane V∗) or transfected with pRc/CMV-TKT plasmid (lane TKT∗) and were the controls for TKT expression. (B) Phosphorylation status of TKT in Zta-expressing stable clones. RHEK vector control (lane V) and RHEK-Z2 (lane Z2) cells were immunoprecipitated with antiphosphotyrosine antibody (4G10) or irrelevant antibody control (con), subjected to SDS-PAGE (10% polyacrylamide), and blotted with anti-TKT antibody as described in Materials and Methods. The numbers to the left indicate the molecular mass markers in kilodaltons. (C) Expression of the TKT downstream effector MMP-1 transcripts detected by RT-PCR. RNA extracted from RHEK parental (lane P), vector control (lane V), and RHEK-Z2 (lane Z2) cells was subjected to RT and amplified by PCR using Zta-, TKT-, MMP-1-, and hypoxanthine phosphoribosyltransferase (HP)-specific primers. The reaction without reverse transcriptase served as a control to rule out the possibility of contamination with genomic DNA. The reaction detecting HP transcripts was an internal control for RNA quality and amounts.

    Article Snippet: For qualitative PCR amplification, 25-μl reaction mixtures containing 2.5 μl of cDNA, 0.2 mM dNTP, 0.2 μM primers, 1× PCR buffer, and 1 U of Dynazyme II DNA polymerase (Finnzymes, Oy, Finland) were amplified for 25 or 40 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min.

    Techniques: Expressing, Western Blot, Incubation, Labeling, Transfection, Plasmid Preparation, Clone Assay, Immunoprecipitation, SDS Page, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

    Insulin-induced interaction between IRK and Neu1 on the cell surface. A : Colocalization of IRK and Neu1 on the plasma membrane. HEK cells expressing Neu1-YFP and IRK-hRluc chimeric proteins were fixed with paraformaldehyde, permeabilized by Triton X-100, and costained with mouse anti-insulin receptor β-chain (Cell Signaling) (1:400) and rabbit anti-Neu1 (1:100) antibodies. Cells were then counterstained with Oregon Green 488–conjugated anti-rabbit IgG antibodies and Texas Red–conjugated goat anti-mouse antibodies (1:1,000; Molecular Probes). Slides were studied on a Zeiss LSM510 inverted confocal microscope. B : Detection of interaction between IRK and Neu1 by BRET. HEK cells expressing Neu1-YFP and IRK-hRluc ( left ) or YFP and IRK-hRluc ( right ) were treated with coelenterazine H in the absence or in the presence of 5 and 50 nmol/L insulin, and the BRET signal was calculated as the ratio of light intensity emitted by IRK-hRluc (530 nm) and Neu1-YFP (480 nm). In the absence of insulin, only the background levels of energy transfer between Neu1-YFP and IRK-hRluc are observed, whereas incubation of cells with insulin at physiological concentration of 5 nmol/L results in the appearance of BRET signal. C : Coimmunoprecipitation of IRK and Neu1. Hek293T cells overexpressing IRK-hRluc, Neu1-YFP, and CathA plasmids were treated or not with 20 nmol/L insulin for 15 min, washed with ice-cold PBS, and lysed in RIPA buffer containing dithiobis(succinimidyl propionate) cross-linker. After removal of cell debris by centrifugation, supernatants were subjected to immunoprecipitation using anti-insulin receptor β-chain antibodies. Proteins in cell lysate (L), supernatant of cell extract (S), protein A agarose washes (W), and eluate (E) were analyzed by Western blotting using anti-insulin receptor β-chain and anti-Neu1 antibodies.

    Journal: Diabetes

    Article Title: Positive Regulation of Insulin Signaling by Neuraminidase 1

    doi: 10.2337/db12-1825

    Figure Lengend Snippet: Insulin-induced interaction between IRK and Neu1 on the cell surface. A : Colocalization of IRK and Neu1 on the plasma membrane. HEK cells expressing Neu1-YFP and IRK-hRluc chimeric proteins were fixed with paraformaldehyde, permeabilized by Triton X-100, and costained with mouse anti-insulin receptor β-chain (Cell Signaling) (1:400) and rabbit anti-Neu1 (1:100) antibodies. Cells were then counterstained with Oregon Green 488–conjugated anti-rabbit IgG antibodies and Texas Red–conjugated goat anti-mouse antibodies (1:1,000; Molecular Probes). Slides were studied on a Zeiss LSM510 inverted confocal microscope. B : Detection of interaction between IRK and Neu1 by BRET. HEK cells expressing Neu1-YFP and IRK-hRluc ( left ) or YFP and IRK-hRluc ( right ) were treated with coelenterazine H in the absence or in the presence of 5 and 50 nmol/L insulin, and the BRET signal was calculated as the ratio of light intensity emitted by IRK-hRluc (530 nm) and Neu1-YFP (480 nm). In the absence of insulin, only the background levels of energy transfer between Neu1-YFP and IRK-hRluc are observed, whereas incubation of cells with insulin at physiological concentration of 5 nmol/L results in the appearance of BRET signal. C : Coimmunoprecipitation of IRK and Neu1. Hek293T cells overexpressing IRK-hRluc, Neu1-YFP, and CathA plasmids were treated or not with 20 nmol/L insulin for 15 min, washed with ice-cold PBS, and lysed in RIPA buffer containing dithiobis(succinimidyl propionate) cross-linker. After removal of cell debris by centrifugation, supernatants were subjected to immunoprecipitation using anti-insulin receptor β-chain antibodies. Proteins in cell lysate (L), supernatant of cell extract (S), protein A agarose washes (W), and eluate (E) were analyzed by Western blotting using anti-insulin receptor β-chain and anti-Neu1 antibodies.

    Article Snippet: After 48 h, the cells were treated or not with 20 mmol/L insulin for 15 min, washed with ice-cold PBS, collected and resuspended in RIPA buffer containing 0.2 mmol/L dithiobis(succinimidyl propionate), vortexed, placed on ice for 30 min, cleared by centrifugation (20 min at 12,000g and 4°C), and incubated with anti-insulin receptor β-chain antibodies (Cell Signaling) for 4 h at 4°C.

    Techniques: Expressing, Microscopy, Bioluminescence Resonance Energy Transfer, Incubation, Concentration Assay, Centrifugation, Immunoprecipitation, Western Blot

    Confirmation of glpD disruption. A ) Southern blot analysis confirms the disruption of glpD . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI and blotted on a nylon membrane. Digoxygenein-11-dUTP labeled bb0243 probe was utilized to visualize glpD . Migration positions of DNA molecular size markers are indicated on the left. B ) glpD disruption occurred via by a double crossover insertion of flgB - aadA . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI or EcoRI, which would be specific for the proximal or distal bb0243 flanking chromosomal regions, respectively, and blotted to a nylon membrane. Blots were developed with a digoxygenein-11-dUTP-labeled aadA probe. A schematic diagram indicates the sizes of the fragments expected to contain flgB - aadA . Migration positions of DNA molecular size markers are indicated on the left of each panel.

    Journal: PLoS Pathogens

    Article Title: Borrelia burgdorferi Requires Glycerol for Maximum Fitness During The Tick Phase of the Enzootic Cycle

    doi: 10.1371/journal.ppat.1002102

    Figure Lengend Snippet: Confirmation of glpD disruption. A ) Southern blot analysis confirms the disruption of glpD . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI and blotted on a nylon membrane. Digoxygenein-11-dUTP labeled bb0243 probe was utilized to visualize glpD . Migration positions of DNA molecular size markers are indicated on the left. B ) glpD disruption occurred via by a double crossover insertion of flgB - aadA . Whole genomic DNA from B31-A3 and glpD disruption mutants CP176, CP177, CP257 were digested with BamHI or EcoRI, which would be specific for the proximal or distal bb0243 flanking chromosomal regions, respectively, and blotted to a nylon membrane. Blots were developed with a digoxygenein-11-dUTP-labeled aadA probe. A schematic diagram indicates the sizes of the fragments expected to contain flgB - aadA . Migration positions of DNA molecular size markers are indicated on the left of each panel.

    Article Snippet: B. burgdorferi DNA was fragmented by incubation with 4.5 units of BamHI in 1× buffer B (Fermentas, Glen Burnie, MD) or EcoRI in buffer EcoRI (Fermentas) overnight at 37°C.

    Techniques: Southern Blot, Labeling, Migration

    PRDM9, EWSR1, and CDYL coimmunoprecipitate each other and chromosomal axis/synaptonemal complex proteins from wild-type 14-dpp spermatocytes. (A) PRDM9 and its interactors coimmunoprecipitate each other. Left, coIP with anti-PRDM9; middle, coIP with anti-EWSR1; right, coIP with CDYL. Lane 1, input, 10 µg (2.5 µg in the CDYL and EHMT2 blots); lane 2, IgG, 10 µg, coIP with nonimmune IgG; lanes 2 and 3, αPRDM9, αEWSR1, and αCDYL, 10 µg, coIP with the respective antibody, either nontreated (lane 3) or DNase treated (lane 4). The antibodies used to detect specific proteins on Western blots are shown on the left. (B) PRDM9 and its interactors also interact with chromosomal axis/SC proteins. coIP with the same antibodies used in A but probed with antibodies against chromosome axis/SC proteins as marked on the left. (C) Reciprocal coIP shows that SC proteins coimmunoprecipitate PRDM9 and its interactors. coIP with SYCP3 (left) or SYCP1 (right). The antibodies used for detection on Western blots are on the left. (D) Interaction between REC8 and PRDM9 in cultured cells. REC8 was cloned under a V5 tag, and PRDM9 was cloned under a FLAG tag. The two proteins were either coexpressed or separately expressed in HEK 293 cells. Specific interaction between the two proteins was detected in extracts of cells in which the two proteins were coexpressed (Co, left) but not in mixed extracts of cells in which the two proteins were expressed separately (Mix, right). Note that the interacting band is the middle of three bands, indicating that it is a phosphorylated form.

    Journal: Molecular Biology of the Cell

    Article Title: PRDM9 interactions with other proteins provide a link between recombination hotspots and the chromosomal axis in meiosis

    doi: 10.1091/mbc.E16-09-0686

    Figure Lengend Snippet: PRDM9, EWSR1, and CDYL coimmunoprecipitate each other and chromosomal axis/synaptonemal complex proteins from wild-type 14-dpp spermatocytes. (A) PRDM9 and its interactors coimmunoprecipitate each other. Left, coIP with anti-PRDM9; middle, coIP with anti-EWSR1; right, coIP with CDYL. Lane 1, input, 10 µg (2.5 µg in the CDYL and EHMT2 blots); lane 2, IgG, 10 µg, coIP with nonimmune IgG; lanes 2 and 3, αPRDM9, αEWSR1, and αCDYL, 10 µg, coIP with the respective antibody, either nontreated (lane 3) or DNase treated (lane 4). The antibodies used to detect specific proteins on Western blots are shown on the left. (B) PRDM9 and its interactors also interact with chromosomal axis/SC proteins. coIP with the same antibodies used in A but probed with antibodies against chromosome axis/SC proteins as marked on the left. (C) Reciprocal coIP shows that SC proteins coimmunoprecipitate PRDM9 and its interactors. coIP with SYCP3 (left) or SYCP1 (right). The antibodies used for detection on Western blots are on the left. (D) Interaction between REC8 and PRDM9 in cultured cells. REC8 was cloned under a V5 tag, and PRDM9 was cloned under a FLAG tag. The two proteins were either coexpressed or separately expressed in HEK 293 cells. Specific interaction between the two proteins was detected in extracts of cells in which the two proteins were coexpressed (Co, left) but not in mixed extracts of cells in which the two proteins were expressed separately (Mix, right). Note that the interacting band is the middle of three bands, indicating that it is a phosphorylated form.

    Article Snippet: For the DNase-treated coIP samples, 100 µl of DNase buffer and 20 U DNase (Ambion) were added, and the samples were incubated for 1 h at room temperature.

    Techniques: Co-Immunoprecipitation Assay, Western Blot, Cell Culture, Clone Assay, FLAG-tag

    Analysis of the Mur34 binding site by DNase I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.

    Journal: PLoS ONE

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471

    doi: 10.1371/journal.pone.0076068

    Figure Lengend Snippet: Analysis of the Mur34 binding site by DNase I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.

    Article Snippet: For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min.

    Techniques: Binding Assay, Footprinting, Labeling, Sequencing, Purification

    Gene expression analysis of the  mur  genes. (A) Transcription analysis of intergenic region of the selected  mur  genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions.  mur10 ← mur11  means that the detected region between  mur10  and  mur11 , and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of  mur11  and  mur27  for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.

    Journal: PLoS ONE

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471

    doi: 10.1371/journal.pone.0076068

    Figure Lengend Snippet: Gene expression analysis of the mur genes. (A) Transcription analysis of intergenic region of the selected mur genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions. mur10 ← mur11 means that the detected region between mur10 and mur11 , and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of mur11 and mur27 for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.

    Article Snippet: For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min.

    Techniques: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction, Negative Control, Incubation

    Cellular function reinstitution of genome-edited RBCs. a For cellular function study, in vitro RBC differentiation of cloned erythroid progenitor cells was induced. Nuclei of the resultant cells were initially stained with Hoechst dye and examined under microscope, demonstrating induced in vitro RBC differentiation and maturation. b Cytospin of resultant cells with Wright-Giemsa staining confirmed presence of mature RBCs (no nuclei), RBC with nucleus contraction, and nucleated RBC. c Cell sickling assays of in vitro differentiated RBCs with or without genome editing, and RBCs from the SCD, SCT, and healthy donor blood demonstrated that CRISPR genome editing of patient HSPCs resulted in function reinstitution of the cloned offspring RBCs, which became more resistant to hypoxia. d Time course of sickling cell formation (%). The data are from the average of three clones

    Journal: Journal of Hematology & Oncology

    Article Title: Cellular function reinstitution of offspring red blood cells cloned from the sickle cell disease patient blood post CRISPR genome editing

    doi: 10.1186/s13045-017-0489-9

    Figure Lengend Snippet: Cellular function reinstitution of genome-edited RBCs. a For cellular function study, in vitro RBC differentiation of cloned erythroid progenitor cells was induced. Nuclei of the resultant cells were initially stained with Hoechst dye and examined under microscope, demonstrating induced in vitro RBC differentiation and maturation. b Cytospin of resultant cells with Wright-Giemsa staining confirmed presence of mature RBCs (no nuclei), RBC with nucleus contraction, and nucleated RBC. c Cell sickling assays of in vitro differentiated RBCs with or without genome editing, and RBCs from the SCD, SCT, and healthy donor blood demonstrated that CRISPR genome editing of patient HSPCs resulted in function reinstitution of the cloned offspring RBCs, which became more resistant to hypoxia. d Time course of sickling cell formation (%). The data are from the average of three clones

    Article Snippet: Briefly, 2–3 mL of the blood was collected, transferred to a 50-mL conical tube, and RBCs were lysed by incubation in 20 mL of RBC lysis buffer for 10 min (Biolegend, San Diego, CA).

    Techniques: Cell Function Assay, In Vitro, Clone Assay, Staining, Microscopy, CRISPR

    Trib1 overexpression in B cells induces a reduction in the production of secreted form of IgG1. Total splenocytes from Trib1-ROSA and Trib1-ROSA Mb1Cre mice were stimulated with LPS/IL-4 for 72 h in vitro [with (+) or without (Ø) the addition of a protein transport inhibitor “GolgiStop™” for the last 8 h of culture], then stained for intracytoplasmic IgG1 after a step of membrane Ig blocking using an anti-murine IgG antibody, a step of fixation and permeabilization, and finally were analyzed by flow cytometry. (A) Percentage of B220 + B cells stained for intracellular IgG1. (B) MFI of intracellular IgG1 staining on B220 + B cells. (C) A representative sample of each condition is shown. Each dot represents the result for one animal.

    Journal: Frontiers in Immunology

    Article Title: Trib1 Is Overexpressed in Systemic Lupus Erythematosus, While It Regulates Immunoglobulin Production in Murine B Cells

    doi: 10.3389/fimmu.2018.00373

    Figure Lengend Snippet: Trib1 overexpression in B cells induces a reduction in the production of secreted form of IgG1. Total splenocytes from Trib1-ROSA and Trib1-ROSA Mb1Cre mice were stimulated with LPS/IL-4 for 72 h in vitro [with (+) or without (Ø) the addition of a protein transport inhibitor “GolgiStop™” for the last 8 h of culture], then stained for intracytoplasmic IgG1 after a step of membrane Ig blocking using an anti-murine IgG antibody, a step of fixation and permeabilization, and finally were analyzed by flow cytometry. (A) Percentage of B220 + B cells stained for intracellular IgG1. (B) MFI of intracellular IgG1 staining on B220 + B cells. (C) A representative sample of each condition is shown. Each dot represents the result for one animal.

    Article Snippet: Cells were then permeabilized with the permeabilization buffer from Fixation/Permeabilization kit (eBioscience) and incubated for 30 min at room temperature in the dark with anti-mouse IgG1 (PE, Southern Biotech) and washed before acquisition by a cytometer.

    Techniques: Over Expression, Mouse Assay, In Vitro, Staining, Blocking Assay, Flow Cytometry, Cytometry

    Expression of PD-1/CTLA-4 in CD4 + and CD8 + T cells PBMC from HD and PBC patients, NILs and TILs were stained for CD3, CD4 and PD-1 surface markers. After fixation and permeabilization, cells were stained for intracellular CTLA-4. Live cells were gated using Fixable Viability Dye 660. Representative flow cytometric plots showing PD-1 and CTLA-4 co-expression in CD4 + T cells ( A ) and whisker plots ( B ) showing differences in their expression in HD-PBMC, PBC-PBMC, NILs and TILs. ( C ). Pie charts show the relative percentages of PD-1 and CTLA-4 co-expression in CD4 + T cells. ( D ). Representative flow cytometric plots for co-expression of PD-1/CTLA-4 in CD8 + cells in NILs and TILs are shown.

    Journal: Oncotarget

    Article Title: Preferential accumulation of regulatory T cells with highly immunosuppressive characteristics in breast tumor microenvironment

    doi: 10.18632/oncotarget.16565

    Figure Lengend Snippet: Expression of PD-1/CTLA-4 in CD4 + and CD8 + T cells PBMC from HD and PBC patients, NILs and TILs were stained for CD3, CD4 and PD-1 surface markers. After fixation and permeabilization, cells were stained for intracellular CTLA-4. Live cells were gated using Fixable Viability Dye 660. Representative flow cytometric plots showing PD-1 and CTLA-4 co-expression in CD4 + T cells ( A ) and whisker plots ( B ) showing differences in their expression in HD-PBMC, PBC-PBMC, NILs and TILs. ( C ). Pie charts show the relative percentages of PD-1 and CTLA-4 co-expression in CD4 + T cells. ( D ). Representative flow cytometric plots for co-expression of PD-1/CTLA-4 in CD8 + cells in NILs and TILs are shown.

    Article Snippet: Following incubation, cells were washed twice with staining solution and fixed/permeabilized using fixation/permeabilization buffer (eBioscience) at 4°C for 45 min. After two washes with permeabilization wash buffer, cells were blocked using mouse serum (M5905, Sigma-Aldrich) and rat serum (R9759, Sigma-Aldrich) for 10 min and stained with CTLA-4-PerCP-eFluor® 710 (46-1529-42, eBioscience), FoxP3-PE-Cy7 (25-4776-42, eBioscience) and Helios-FITC (137214, BioLegend) antibodies for another 30 minutes at 4°C.

    Techniques: Expressing, Staining, Flow Cytometry, Whisker Assay

    Detection of intranuclear markers using the adapted BD cytofix/cytoperm protocol. Cells from healthy donors were incubated with antibodies targeting cell surface antigens and then split into 5 for the following conditions: the “surface staining only” conditions (CS), and fixation and permeabilization using either BD cytofix/cytoperm buffer (ICSb), eBioscience fixation and permeabilization buffer (INSb 1), Maxpar NASB (INSb 2) and PFA/methanol (INSb 3). Next cells were labelled with a mix of antibodies targeting intranuclear markers. Data compare the frequencies of Treg cells (CD25hiFoxP3+), Tfh cells (CD4+BCL6+), Th17 cells (CD4+RoryT+) and CD8+Tbet+ cells between the various permeabilization conditions. The concentrations of antibodies used are as follow: FoxP3 (0.3 mg/ml), BCL6 (0.8 mg/ml), RoryT (0.6 mg/ml) and Tbet (0.3 mg/ml). Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p

    Journal: PLoS ONE

    Article Title: Analysis of cell surface and intranuclear markers on non-stimulated human PBMC using mass cytometry

    doi: 10.1371/journal.pone.0194593

    Figure Lengend Snippet: Detection of intranuclear markers using the adapted BD cytofix/cytoperm protocol. Cells from healthy donors were incubated with antibodies targeting cell surface antigens and then split into 5 for the following conditions: the “surface staining only” conditions (CS), and fixation and permeabilization using either BD cytofix/cytoperm buffer (ICSb), eBioscience fixation and permeabilization buffer (INSb 1), Maxpar NASB (INSb 2) and PFA/methanol (INSb 3). Next cells were labelled with a mix of antibodies targeting intranuclear markers. Data compare the frequencies of Treg cells (CD25hiFoxP3+), Tfh cells (CD4+BCL6+), Th17 cells (CD4+RoryT+) and CD8+Tbet+ cells between the various permeabilization conditions. The concentrations of antibodies used are as follow: FoxP3 (0.3 mg/ml), BCL6 (0.8 mg/ml), RoryT (0.6 mg/ml) and Tbet (0.3 mg/ml). Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p

    Article Snippet: We observed median percentages of 2.7%, 0.2%, 0.1% and 38% respectively for Tregs, Th17 cells, Tfh cells and CD8+Tbet+ cells with eBioscience Permeabilization buffer versus 2.2%, 0.2%, 0.05% and 30% respectively for BD cytofix/cytoperm buffer ( ).

    Techniques: Incubation, Staining

    T regulatory cells are increased in infants On d14 p.i., the tracheobronchial lymph nodes (TBLN) (A), lung (B) and spleen (C) were isolated and processed to yield single cell suspensions. S pleens were also isolated from non-infected animals (C) . Cells were stained for CD4 + and FoxP3 + . Averaged data shown are the percentage of FoxP3-expressing cells within the CD4 + population. Significance at each timepoint was determined using a student’s t test. *p≤0.05, **p ≤ 0.01.

    Journal: Virology

    Article Title: Nonhuman primate infants have an impaired respiratory but not systemic IgG antibody response following influenza virus infection

    doi: 10.1016/j.virol.2014.12.007

    Figure Lengend Snippet: T regulatory cells are increased in infants On d14 p.i., the tracheobronchial lymph nodes (TBLN) (A), lung (B) and spleen (C) were isolated and processed to yield single cell suspensions. S pleens were also isolated from non-infected animals (C) . Cells were stained for CD4 + and FoxP3 + . Averaged data shown are the percentage of FoxP3-expressing cells within the CD4 + population. Significance at each timepoint was determined using a student’s t test. *p≤0.05, **p ≤ 0.01.

    Article Snippet: Following washing cells were permeabilized using BioLegend FOXP3 Fix/Perm and FOXP3 Perm buffer (BioLegend) followed by incubation with anti-FoxP3 antibody (Clone 206D, BioLegend).

    Techniques: Isolation, Infection, Staining, Expressing

    Increased frequency and expression of FOXP3 in low dose cultures correlates with increased suppressive function

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

    Article Title: Low dose antigen promotes induction of FOXP3 in human CD4+ T cells

    doi: 10.4049/jimmunol.1003880

    Figure Lengend Snippet: Increased frequency and expression of FOXP3 in low dose cultures correlates with increased suppressive function

    Article Snippet: Briefly, following over-night incubation with BrdU cells were fixed and permeabilized using the Biolegend FOXP3 fixation buffer and then the BD Cytofix/Cytoperm and BD Cytoperm Plus buffers.

    Techniques: Expressing

    The frequency of FOXP3+ cells in aTreg is not maintained upon re-stimulation

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

    Article Title: Low dose antigen promotes induction of FOXP3 in human CD4+ T cells

    doi: 10.4049/jimmunol.1003880

    Figure Lengend Snippet: The frequency of FOXP3+ cells in aTreg is not maintained upon re-stimulation

    Article Snippet: Briefly, following over-night incubation with BrdU cells were fixed and permeabilized using the Biolegend FOXP3 fixation buffer and then the BD Cytofix/Cytoperm and BD Cytoperm Plus buffers.

    Techniques:

    Lower doses of antigen promote an increased frequency of FOXP3 + cells in influenza specific T cell populations

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

    Article Title: Low dose antigen promotes induction of FOXP3 in human CD4+ T cells

    doi: 10.4049/jimmunol.1003880

    Figure Lengend Snippet: Lower doses of antigen promote an increased frequency of FOXP3 + cells in influenza specific T cell populations

    Article Snippet: Briefly, following over-night incubation with BrdU cells were fixed and permeabilized using the Biolegend FOXP3 fixation buffer and then the BD Cytofix/Cytoperm and BD Cytoperm Plus buffers.

    Techniques:

    FOXP3 + T cells generated in high and low dose cultures proliferate at equivalent rates

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

    Article Title: Low dose antigen promotes induction of FOXP3 in human CD4+ T cells

    doi: 10.4049/jimmunol.1003880

    Figure Lengend Snippet: FOXP3 + T cells generated in high and low dose cultures proliferate at equivalent rates

    Article Snippet: Briefly, following over-night incubation with BrdU cells were fixed and permeabilized using the Biolegend FOXP3 fixation buffer and then the BD Cytofix/Cytoperm and BD Cytoperm Plus buffers.

    Techniques: Generated

    Lower doses of antigen favor an increased frequency of FOXP3 + cells in islet antigen specific T cells

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

    Article Title: Low dose antigen promotes induction of FOXP3 in human CD4+ T cells

    doi: 10.4049/jimmunol.1003880

    Figure Lengend Snippet: Lower doses of antigen favor an increased frequency of FOXP3 + cells in islet antigen specific T cells

    Article Snippet: Briefly, following over-night incubation with BrdU cells were fixed and permeabilized using the Biolegend FOXP3 fixation buffer and then the BD Cytofix/Cytoperm and BD Cytoperm Plus buffers.

    Techniques:

    TGFβ and TCR both contribute to FOXP3 expression upon activation of CD4 T cells

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

    Article Title: Low dose antigen promotes induction of FOXP3 in human CD4+ T cells

    doi: 10.4049/jimmunol.1003880

    Figure Lengend Snippet: TGFβ and TCR both contribute to FOXP3 expression upon activation of CD4 T cells

    Article Snippet: Briefly, following over-night incubation with BrdU cells were fixed and permeabilized using the Biolegend FOXP3 fixation buffer and then the BD Cytofix/Cytoperm and BD Cytoperm Plus buffers.

    Techniques: Expressing, Activation Assay

    Association between lifetime intimate partner violence (IPV) experience and regulatory (CD4 + Foxp3 + CD25 + ) T-cell frequency and phenotype among HIV high-risk women (a) Representative flow diagrams demonstrating frequencies of regulatory (CD4 + Foxp3 + CD25 + ) T-cells in the PBMC of two donors with low versus high reporting of lifetime IPV experience. (b) Summary data from 75 HIV high-risk women demonstrating lifetime IPV experience was not significantly associated with frequency of regulatory (CD4 + FoxP3 + CD25 + ) T-cells in PBMC. (c) Representative flow diagrams depicting frequencies of regulatory (CD4 + FoxP3 + CD25 + ) T-cell phenotypes using CD45RA and HLA-DR markers in the PBMC of two donors with low versus high reporting of lifetime IPV experience. (d) Summary data from 75 HIV high-risk women demonstrating lifetime IPV experience is negatively associated with the frequency of naïve regulatory T-cells (HLA-DR − CD45RA + CD4 + FoxP3 + CD25 + ) in PBMC.

    Journal: Pathogens & immunity

    Article Title: INTIMATE PARTNER VIOLENCE IS ASSOCIATED WITH INCREASED CD4+ T-CELL ACTIVATION AMONG HIV-NEGATIVE HIGH-RISK WOMEN

    doi: 10.20411/pai.v1i1.120

    Figure Lengend Snippet: Association between lifetime intimate partner violence (IPV) experience and regulatory (CD4 + Foxp3 + CD25 + ) T-cell frequency and phenotype among HIV high-risk women (a) Representative flow diagrams demonstrating frequencies of regulatory (CD4 + Foxp3 + CD25 + ) T-cells in the PBMC of two donors with low versus high reporting of lifetime IPV experience. (b) Summary data from 75 HIV high-risk women demonstrating lifetime IPV experience was not significantly associated with frequency of regulatory (CD4 + FoxP3 + CD25 + ) T-cells in PBMC. (c) Representative flow diagrams depicting frequencies of regulatory (CD4 + FoxP3 + CD25 + ) T-cell phenotypes using CD45RA and HLA-DR markers in the PBMC of two donors with low versus high reporting of lifetime IPV experience. (d) Summary data from 75 HIV high-risk women demonstrating lifetime IPV experience is negatively associated with the frequency of naïve regulatory T-cells (HLA-DR − CD45RA + CD4 + FoxP3 + CD25 + ) in PBMC.

    Article Snippet: Cells were then washed once with FACS wash buffer and fixed with 1× fix/perm buffer (Tonbo Biosciences) for 60 minutes at room temperature, then washed again with 1× perm buffer solution and incubated in this buffer solution for 10 minutes, washed again with 1× perm buffer solution, and incubated for 45 minutes with mAb to Foxp3 (clone 206D, Biolegend).

    Techniques: Flow Cytometry

    Association between past-year intimate partner violence (IPV) experience and regulatory (CD4 + Foxp3 + CD25 + ) T-cell frequency and phenotype among HIV high-risk women (a) Representative flow diagrams demonstrating frequencies of regulatory (CD4 + Foxp3 + CD25 + ) T-cells in the PBMC of two donors with low versus high reporting of past-year IPV experience. (b) Summary data from 75 HIV high-risk women demonstrating past-year IPV experience was not significantly associated with frequency of regulatory (CD4 + FoxP3 + CD25 + ) T-cells in PBMC. (c) Representative flow diagrams depicting frequencies of regulatory (CD4 + FoxP3 + CD25 + ) T-cell phenotypes using CD45RA and HLA-DR markers in the PBMC of two donors with low versus high reporting of past-year IPV experience. (d) Summary data from 75 HIV high-risk women demonstrating past-year IPV experience is negatively associated with the frequency of naïve regulatory T-cells (HLA-DR − CD45RA + CD4 + FoxP3 + CD25 + ) in PBMC.

    Journal: Pathogens & immunity

    Article Title: INTIMATE PARTNER VIOLENCE IS ASSOCIATED WITH INCREASED CD4+ T-CELL ACTIVATION AMONG HIV-NEGATIVE HIGH-RISK WOMEN

    doi: 10.20411/pai.v1i1.120

    Figure Lengend Snippet: Association between past-year intimate partner violence (IPV) experience and regulatory (CD4 + Foxp3 + CD25 + ) T-cell frequency and phenotype among HIV high-risk women (a) Representative flow diagrams demonstrating frequencies of regulatory (CD4 + Foxp3 + CD25 + ) T-cells in the PBMC of two donors with low versus high reporting of past-year IPV experience. (b) Summary data from 75 HIV high-risk women demonstrating past-year IPV experience was not significantly associated with frequency of regulatory (CD4 + FoxP3 + CD25 + ) T-cells in PBMC. (c) Representative flow diagrams depicting frequencies of regulatory (CD4 + FoxP3 + CD25 + ) T-cell phenotypes using CD45RA and HLA-DR markers in the PBMC of two donors with low versus high reporting of past-year IPV experience. (d) Summary data from 75 HIV high-risk women demonstrating past-year IPV experience is negatively associated with the frequency of naïve regulatory T-cells (HLA-DR − CD45RA + CD4 + FoxP3 + CD25 + ) in PBMC.

    Article Snippet: Cells were then washed once with FACS wash buffer and fixed with 1× fix/perm buffer (Tonbo Biosciences) for 60 minutes at room temperature, then washed again with 1× perm buffer solution and incubated in this buffer solution for 10 minutes, washed again with 1× perm buffer solution, and incubated for 45 minutes with mAb to Foxp3 (clone 206D, Biolegend).

    Techniques: Flow Cytometry