Structured Review

Bio-Techne corporation gp96
a , Relative expression of ETV3 or ETV6 in blood monocytes, mo-Macs and mo-DCs isolated from peritoneal ascites or generated in vitro (accession nos. GSE102046 and GSE40484 ). a.u., arbitrary units. b – g , Monocytes were cultured with M-CSF, IL-4 and TNF. b , At day 5, mo-Macs and mo-DCs were sorted and lysed for immunoblot analysis. <t>GP96</t> was used as a loading control. Representative results are shown ( n = 5), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). c – f , ETV3 or ETV6 expression was silenced using a lentivirus-containing shRNA. c , e , Protein quantification by immunoblotting after 5 d for ETV3 (c) or ETV6 (e). Actin was used as a loading control. Representative results are shown ( n = 8), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). d , f , Mo-mac and mo-DC differentiation from monocytes after 5 d of ETV3 (d) or ETV6 (f) silencing . One representative donor is shown ( n = 8) and the median ( n = 8 in three independent experiments; paired one-way analysis of variance (ANOVA)). DN, double negative. g , ETV3 or ETV6 mRNA expression was analyzed by RT–qPCR. Each symbol represents an individual donor ( n = 6 in three independent experiments). h , Monocytes were cultured for 3 and 6 h with medium only or combinations of M-CSF, IL-4 and TNF. Each symbol represents an individual donor ( n = 5 in two independent experiments; paired one-way ANOVA). For all panels: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All statistical tests were two sided. For immunoblots, paired samples were derived from the same experiment and processed in parallel.
Gp96, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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gp96 - by Bioz Stars, 2024-04
86/100 stars

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1) Product Images from "ETV3 and ETV6 enable monocyte differentiation into dendritic cells by repressing macrophage fate commitment"

Article Title: ETV3 and ETV6 enable monocyte differentiation into dendritic cells by repressing macrophage fate commitment

Journal: Nature Immunology

doi: 10.1038/s41590-022-01374-0

a , Relative expression of ETV3 or ETV6 in blood monocytes, mo-Macs and mo-DCs isolated from peritoneal ascites or generated in vitro (accession nos. GSE102046 and GSE40484 ). a.u., arbitrary units. b – g , Monocytes were cultured with M-CSF, IL-4 and TNF. b , At day 5, mo-Macs and mo-DCs were sorted and lysed for immunoblot analysis. GP96 was used as a loading control. Representative results are shown ( n = 5), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). c – f , ETV3 or ETV6 expression was silenced using a lentivirus-containing shRNA. c , e , Protein quantification by immunoblotting after 5 d for ETV3 (c) or ETV6 (e). Actin was used as a loading control. Representative results are shown ( n = 8), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). d , f , Mo-mac and mo-DC differentiation from monocytes after 5 d of ETV3 (d) or ETV6 (f) silencing . One representative donor is shown ( n = 8) and the median ( n = 8 in three independent experiments; paired one-way analysis of variance (ANOVA)). DN, double negative. g , ETV3 or ETV6 mRNA expression was analyzed by RT–qPCR. Each symbol represents an individual donor ( n = 6 in three independent experiments). h , Monocytes were cultured for 3 and 6 h with medium only or combinations of M-CSF, IL-4 and TNF. Each symbol represents an individual donor ( n = 5 in two independent experiments; paired one-way ANOVA). For all panels: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All statistical tests were two sided. For immunoblots, paired samples were derived from the same experiment and processed in parallel.
Figure Legend Snippet: a , Relative expression of ETV3 or ETV6 in blood monocytes, mo-Macs and mo-DCs isolated from peritoneal ascites or generated in vitro (accession nos. GSE102046 and GSE40484 ). a.u., arbitrary units. b – g , Monocytes were cultured with M-CSF, IL-4 and TNF. b , At day 5, mo-Macs and mo-DCs were sorted and lysed for immunoblot analysis. GP96 was used as a loading control. Representative results are shown ( n = 5), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). c – f , ETV3 or ETV6 expression was silenced using a lentivirus-containing shRNA. c , e , Protein quantification by immunoblotting after 5 d for ETV3 (c) or ETV6 (e). Actin was used as a loading control. Representative results are shown ( n = 8), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). d , f , Mo-mac and mo-DC differentiation from monocytes after 5 d of ETV3 (d) or ETV6 (f) silencing . One representative donor is shown ( n = 8) and the median ( n = 8 in three independent experiments; paired one-way analysis of variance (ANOVA)). DN, double negative. g , ETV3 or ETV6 mRNA expression was analyzed by RT–qPCR. Each symbol represents an individual donor ( n = 6 in three independent experiments). h , Monocytes were cultured for 3 and 6 h with medium only or combinations of M-CSF, IL-4 and TNF. Each symbol represents an individual donor ( n = 5 in two independent experiments; paired one-way ANOVA). For all panels: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All statistical tests were two sided. For immunoblots, paired samples were derived from the same experiment and processed in parallel.

Techniques Used: Expressing, Isolation, Generated, In Vitro, Cell Culture, Western Blot, shRNA, Quantitative RT-PCR, Derivative Assay

a – d , f , g , Monocytes were cultured with M-CSF, IL-4 and TNF for 3 d. ChIP–seq analysis was performed for ETV3 or ETV6. a , Motif enrichment analysis obtained with HOMER. b , Most enriched motifs. c , Overlap of identified genes for ETV3 or ETV6 immunoprecipitation (IP). Peaks in all gene regions or only in the transcription start site (TSS) region are shown. d , Overlap of DEGs found in RNA-seq and genes identified in ChIP–seq. e , f , Data from RNA-seq analysis. e , GSEA of monocyte, mo-Mac and mo-DC signatures in control (red) versus silenced (blue) samples. NES, normalized enrichment score. f , Heatmap of DEGs belonging to the monocyte, mo-Mac or mo-DC signatures. Samples were ordered by condition and genes were ordered manually. Genes detected in ChIP–seq analysis are emboldened. g , Gene tracks from ChIP–seq analysis for the genomic region of MAFB . h – j , Monocytes were cultured with M-CSF for 5 d. ETV6 was overexpressed using a lentivirus containing an expression plasmid. h , Protein quantification by immunblotting. GP96 was used as a loading control. Representative results are shown ( n = 5 in two independent experiments). Paired samples derive from the same experiment and were processed in parallel. i , Mo-Mac differentiation. One representative donor is shown ( n = 11). j , Percentage of mo-Macs after 5 d ( n = 11 in four independent experiments; paired Student’s t -test: *** P < 0.001). All statistical tests were two sided.
Figure Legend Snippet: a – d , f , g , Monocytes were cultured with M-CSF, IL-4 and TNF for 3 d. ChIP–seq analysis was performed for ETV3 or ETV6. a , Motif enrichment analysis obtained with HOMER. b , Most enriched motifs. c , Overlap of identified genes for ETV3 or ETV6 immunoprecipitation (IP). Peaks in all gene regions or only in the transcription start site (TSS) region are shown. d , Overlap of DEGs found in RNA-seq and genes identified in ChIP–seq. e , f , Data from RNA-seq analysis. e , GSEA of monocyte, mo-Mac and mo-DC signatures in control (red) versus silenced (blue) samples. NES, normalized enrichment score. f , Heatmap of DEGs belonging to the monocyte, mo-Mac or mo-DC signatures. Samples were ordered by condition and genes were ordered manually. Genes detected in ChIP–seq analysis are emboldened. g , Gene tracks from ChIP–seq analysis for the genomic region of MAFB . h – j , Monocytes were cultured with M-CSF for 5 d. ETV6 was overexpressed using a lentivirus containing an expression plasmid. h , Protein quantification by immunblotting. GP96 was used as a loading control. Representative results are shown ( n = 5 in two independent experiments). Paired samples derive from the same experiment and were processed in parallel. i , Mo-Mac differentiation. One representative donor is shown ( n = 11). j , Percentage of mo-Macs after 5 d ( n = 11 in four independent experiments; paired Student’s t -test: *** P < 0.001). All statistical tests were two sided.

Techniques Used: Cell Culture, ChIP-sequencing, Immunoprecipitation, RNA Sequencing Assay, Expressing, Plasmid Preparation


Structured Review

Bio-Techne corporation hsp90b1
Hsp90b1, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
hsp90b1 - by Bioz Stars, 2024-04
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Bio-Techne corporation anti hsp90b1
Anti Hsp90b1, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti hsp90b1/product/Bio-Techne corporation
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti hsp90b1 - by Bioz Stars, 2024-04
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Bio-Techne corporation anti mouse gp96
Expression of gB and <t>GP96‐NT</t> plasmids in vitro and in vivo. 3T3 cells were transfected with pgB, pGP96‐NT or vector with PEI for 48 h, and then, cell lysates were subjected to Western blot analysis using anti‐gB, anti‐GP96 or anti‐β‐actin antibody, respectively. A, In vitro expression of gB and GP96‐NT plasmids. Lane 1: 3T3 cells transfected with pcDNA3.1‐GP96‐NT; lane 2: 3T3 cells transfected with pcDNA3.1 vector; lane 3: 3T3 cells transfected with pcDNA3.1‐gB; lane 4: 3T3 cells transfected with pcDNA3.1 vector. B, Quantification of gB and GP96‐NT expression by densitometry in vitro. Data were from one representative experiment of 3 performed and presented as the mean ± SD, and t test was used, *** P < .001. C, Balb/c mice were intranasally immunized with PEI‐pgB or PEI‐pGP96‐NT vaccines, respectively, and lungs were taken 3 days later for gene expression analysis. Western blot analysis of gB, GP96 or β‐actin expression for lung tissues from immunized mice. Lane 1: Mice immunized with pcDNA3.1‐GP96‐NT; lane 2: mice immunized with pcDNA3.1 vector; lane 3: mice immunized with pcDNA3.1‐gB; lane 4: mice immunized with pcDNA3.1 vector. D, Quantification of gB and GP96‐NT expression by densitometry in vivo. Data were from one representative experiment of 3 performed and presented as the mean ± SD (n = 8), and t test was used,*** P < .001
Anti Mouse Gp96, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 86 stars, based on 1 article reviews
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1) Product Images from "gB co‐immunization with GP96 enhances pulmonary‐resident CD8 T cells and exerts a long‐term defence against MCMV pneumonitis"

Article Title: gB co‐immunization with GP96 enhances pulmonary‐resident CD8 T cells and exerts a long‐term defence against MCMV pneumonitis

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.16065

Expression of gB and GP96‐NT plasmids in vitro and in vivo. 3T3 cells were transfected with pgB, pGP96‐NT or vector with PEI for 48 h, and then, cell lysates were subjected to Western blot analysis using anti‐gB, anti‐GP96 or anti‐β‐actin antibody, respectively. A, In vitro expression of gB and GP96‐NT plasmids. Lane 1: 3T3 cells transfected with pcDNA3.1‐GP96‐NT; lane 2: 3T3 cells transfected with pcDNA3.1 vector; lane 3: 3T3 cells transfected with pcDNA3.1‐gB; lane 4: 3T3 cells transfected with pcDNA3.1 vector. B, Quantification of gB and GP96‐NT expression by densitometry in vitro. Data were from one representative experiment of 3 performed and presented as the mean ± SD, and t test was used, *** P < .001. C, Balb/c mice were intranasally immunized with PEI‐pgB or PEI‐pGP96‐NT vaccines, respectively, and lungs were taken 3 days later for gene expression analysis. Western blot analysis of gB, GP96 or β‐actin expression for lung tissues from immunized mice. Lane 1: Mice immunized with pcDNA3.1‐GP96‐NT; lane 2: mice immunized with pcDNA3.1 vector; lane 3: mice immunized with pcDNA3.1‐gB; lane 4: mice immunized with pcDNA3.1 vector. D, Quantification of gB and GP96‐NT expression by densitometry in vivo. Data were from one representative experiment of 3 performed and presented as the mean ± SD (n = 8), and t test was used,*** P < .001
Figure Legend Snippet: Expression of gB and GP96‐NT plasmids in vitro and in vivo. 3T3 cells were transfected with pgB, pGP96‐NT or vector with PEI for 48 h, and then, cell lysates were subjected to Western blot analysis using anti‐gB, anti‐GP96 or anti‐β‐actin antibody, respectively. A, In vitro expression of gB and GP96‐NT plasmids. Lane 1: 3T3 cells transfected with pcDNA3.1‐GP96‐NT; lane 2: 3T3 cells transfected with pcDNA3.1 vector; lane 3: 3T3 cells transfected with pcDNA3.1‐gB; lane 4: 3T3 cells transfected with pcDNA3.1 vector. B, Quantification of gB and GP96‐NT expression by densitometry in vitro. Data were from one representative experiment of 3 performed and presented as the mean ± SD, and t test was used, *** P < .001. C, Balb/c mice were intranasally immunized with PEI‐pgB or PEI‐pGP96‐NT vaccines, respectively, and lungs were taken 3 days later for gene expression analysis. Western blot analysis of gB, GP96 or β‐actin expression for lung tissues from immunized mice. Lane 1: Mice immunized with pcDNA3.1‐GP96‐NT; lane 2: mice immunized with pcDNA3.1 vector; lane 3: mice immunized with pcDNA3.1‐gB; lane 4: mice immunized with pcDNA3.1 vector. D, Quantification of gB and GP96‐NT expression by densitometry in vivo. Data were from one representative experiment of 3 performed and presented as the mean ± SD (n = 8), and t test was used,*** P < .001

Techniques Used: Expressing, In Vitro, In Vivo, Transfection, Plasmid Preparation, Western Blot, Vaccines


Structured Review

Bio-Techne corporation hsp90b1
GRP78 is a novel binding partner of PD-L1. A. Schematic of the strategy by using these tools to identify potential heat shock proteins to affect the stability of PD-L1. B. Co-immunoprecipitation of western blot was shown to determine the interaction of GRP78 and PD-L1 in MDA-MB-231 and BT-549 cells. C. Co-immunoprecipitation of western blot was shown to determine the interaction of GRP78 and PD-L1. Quantification of indicated proteins were adjusted by GAPDH. D. Flow cytometric analysis of the effect of thapsigargin (16 hours) on surface GRP78 in BT549 cells. E. BT-549 cells treated with or without thapsigargin (16 hours) and immunostaining of BT-549 cells with antibodies against GRP78 and PD-L1. Scale bar, 100 µm. F. GRP78 interacts with PD-L1. Representative images of immunofluorescence staining of GRP78 and PD-L1 interaction in ER region in BT-549 cells treated with thapsigargin (16 hours) by Duo-link assay. The red dots (GRP78/PD-L1 interaction) indicate their interaction. Green fluorescence <t>(HSP90B1)</t> was used as ER marker, and DAPI as a nuclear marker.
Hsp90b1, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The stabilization of PD-L1 by the endoplasmic reticulum stress protein GRP78 in triple-negative breast cancer"

Article Title: The stabilization of PD-L1 by the endoplasmic reticulum stress protein GRP78 in triple-negative breast cancer

Journal: American Journal of Cancer Research

doi:

GRP78 is a novel binding partner of PD-L1. A. Schematic of the strategy by using these tools to identify potential heat shock proteins to affect the stability of PD-L1. B. Co-immunoprecipitation of western blot was shown to determine the interaction of GRP78 and PD-L1 in MDA-MB-231 and BT-549 cells. C. Co-immunoprecipitation of western blot was shown to determine the interaction of GRP78 and PD-L1. Quantification of indicated proteins were adjusted by GAPDH. D. Flow cytometric analysis of the effect of thapsigargin (16 hours) on surface GRP78 in BT549 cells. E. BT-549 cells treated with or without thapsigargin (16 hours) and immunostaining of BT-549 cells with antibodies against GRP78 and PD-L1. Scale bar, 100 µm. F. GRP78 interacts with PD-L1. Representative images of immunofluorescence staining of GRP78 and PD-L1 interaction in ER region in BT-549 cells treated with thapsigargin (16 hours) by Duo-link assay. The red dots (GRP78/PD-L1 interaction) indicate their interaction. Green fluorescence (HSP90B1) was used as ER marker, and DAPI as a nuclear marker.
Figure Legend Snippet: GRP78 is a novel binding partner of PD-L1. A. Schematic of the strategy by using these tools to identify potential heat shock proteins to affect the stability of PD-L1. B. Co-immunoprecipitation of western blot was shown to determine the interaction of GRP78 and PD-L1 in MDA-MB-231 and BT-549 cells. C. Co-immunoprecipitation of western blot was shown to determine the interaction of GRP78 and PD-L1. Quantification of indicated proteins were adjusted by GAPDH. D. Flow cytometric analysis of the effect of thapsigargin (16 hours) on surface GRP78 in BT549 cells. E. BT-549 cells treated with or without thapsigargin (16 hours) and immunostaining of BT-549 cells with antibodies against GRP78 and PD-L1. Scale bar, 100 µm. F. GRP78 interacts with PD-L1. Representative images of immunofluorescence staining of GRP78 and PD-L1 interaction in ER region in BT-549 cells treated with thapsigargin (16 hours) by Duo-link assay. The red dots (GRP78/PD-L1 interaction) indicate their interaction. Green fluorescence (HSP90B1) was used as ER marker, and DAPI as a nuclear marker.

Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, Immunostaining, Immunofluorescence, Staining, Fluorescence, Marker


Structured Review

Bio-Techne corporation hsp90b1
GRP78 is a novel binding partner of PD-L1. A. Schematic of the strategy by using these tools to identify potential heat shock proteins to affect the stability of PD-L1. B. Co-immunoprecipitation of western blot was shown to determine the interaction of GRP78 and PD-L1 in MDA-MB-231 and BT-549 cells. C. Co-immunoprecipitation of western blot was shown to determine the interaction of GRP78 and PD-L1. Quantification of indicated proteins were adjusted by GAPDH. D. Flow cytometric analysis of the effect of thapsigargin (16 hours) on surface GRP78 in BT549 cells. E. BT-549 cells treated with or without thapsigargin (16 hours) and immunostaining of BT-549 cells with antibodies against GRP78 and PD-L1. Scale bar, 100 µm. F. GRP78 interacts with PD-L1. Representative images of immunofluorescence staining of GRP78 and PD-L1 interaction in ER region in BT-549 cells treated with thapsigargin (16 hours) by Duo-link assay. The red dots (GRP78/PD-L1 interaction) indicate their interaction. Green fluorescence <t>(HSP90B1)</t> was used as ER marker, and DAPI as a nuclear marker.
Hsp90b1, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/hsp90b1/product/Bio-Techne corporation
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
hsp90b1 - by Bioz Stars, 2024-04
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1) Product Images from "The stabilization of PD-L1 by the endoplasmic reticulum stress protein GRP78 in triple-negative breast cancer"

Article Title: The stabilization of PD-L1 by the endoplasmic reticulum stress protein GRP78 in triple-negative breast cancer

Journal: American Journal of Cancer Research

doi:

GRP78 is a novel binding partner of PD-L1. A. Schematic of the strategy by using these tools to identify potential heat shock proteins to affect the stability of PD-L1. B. Co-immunoprecipitation of western blot was shown to determine the interaction of GRP78 and PD-L1 in MDA-MB-231 and BT-549 cells. C. Co-immunoprecipitation of western blot was shown to determine the interaction of GRP78 and PD-L1. Quantification of indicated proteins were adjusted by GAPDH. D. Flow cytometric analysis of the effect of thapsigargin (16 hours) on surface GRP78 in BT549 cells. E. BT-549 cells treated with or without thapsigargin (16 hours) and immunostaining of BT-549 cells with antibodies against GRP78 and PD-L1. Scale bar, 100 µm. F. GRP78 interacts with PD-L1. Representative images of immunofluorescence staining of GRP78 and PD-L1 interaction in ER region in BT-549 cells treated with thapsigargin (16 hours) by Duo-link assay. The red dots (GRP78/PD-L1 interaction) indicate their interaction. Green fluorescence (HSP90B1) was used as ER marker, and DAPI as a nuclear marker.
Figure Legend Snippet: GRP78 is a novel binding partner of PD-L1. A. Schematic of the strategy by using these tools to identify potential heat shock proteins to affect the stability of PD-L1. B. Co-immunoprecipitation of western blot was shown to determine the interaction of GRP78 and PD-L1 in MDA-MB-231 and BT-549 cells. C. Co-immunoprecipitation of western blot was shown to determine the interaction of GRP78 and PD-L1. Quantification of indicated proteins were adjusted by GAPDH. D. Flow cytometric analysis of the effect of thapsigargin (16 hours) on surface GRP78 in BT549 cells. E. BT-549 cells treated with or without thapsigargin (16 hours) and immunostaining of BT-549 cells with antibodies against GRP78 and PD-L1. Scale bar, 100 µm. F. GRP78 interacts with PD-L1. Representative images of immunofluorescence staining of GRP78 and PD-L1 interaction in ER region in BT-549 cells treated with thapsigargin (16 hours) by Duo-link assay. The red dots (GRP78/PD-L1 interaction) indicate their interaction. Green fluorescence (HSP90B1) was used as ER marker, and DAPI as a nuclear marker.

Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, Immunostaining, Immunofluorescence, Staining, Fluorescence, Marker


Structured Review

Bio-Techne corporation 143 hsp90b1
143 Hsp90b1, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/143 hsp90b1/product/Bio-Techne corporation
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
143 hsp90b1 - by Bioz Stars, 2024-04
86/100 stars

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Structured Review

Bio-Techne corporation hsp90b1
(A) BT-549 cells were treated with metformin (5 mM) for the indicated time. Detection of endogenous AMPKα and PD-L1 binding (red dots) by Duolink II assay. Three different positions were randomly selected at each point, and the number of red dots were divided by the number of nuclei. Data represent mean ± SD. n = 3. *P, 0.01~0.05, **P, 0.001~0.01, and #P, < 0.001, Student’s t test. Scale bar, 20 μm. Right, MDA-MB-231 WT, PD-L1 KO and AMPKα KO cells were used as negative controls. Scale bar, 25 μm. (B) MDA-MB-231 cells were cultured for 6 hr with or without metformin (5 mM) and MG132 (10 μM). Endogenous PD-L1 and AMPKα were immunoprecipitated and their binding was analyzed with immunoblotting. (C) In vitro kinase activity of AMPK toward PD-L1 with 32P-labeled ATP. (D) Kinetics of PD-L1 phosphorylation by AMPK. Acetyl-CoA carboxylase (ACC) was used as positive control. Km and Vmax were calculated using the Michaelis-Menten equation. (E) In vitro phosphorylation assay and phospho-tag gel shifting assay. W, PD-L1/WT. A, PD-L1/S195A. (F) PD-L1/S195 phosphorylation was examined using anti-PDL1/S195-p antibody at different time points after metformin (5 mM) treatment. (G) Western blot analysis of MDA-MB-231 WT and AMPKα KO cells after metformin treatment (5 mM) for 8 hr. Endogenous PD-L1 purified by IP was subjected to immunoblotting with PDL1 S195-p antibody after PNGase F reaction. (H) PD-L1 and AMPK subcellular localization of MDA-MB-231 WT and AMPKα KO cells. (I) Trypsin digestion of ER fractions with or without permeabilization. (J, K) BT-549 cells were treated with metformin (5 mM) for 3 hr. (J) BT-549 cells were subjected to Duolink II assay combined with immunofluoresence staining using markers for ER <t>(HSP90B1),</t> Golgi (TNG46), and nuclei (Hoechst). Scale bar, 20 μm (inset, 10 μm). (K) Duolink assay with antibodies specific for ECD (Ab205921 and 86744S) and ICD (13684S and GTX104763) of PD-L1. Scale bar, 50 μm (inset, 25 μm). Red dots: AMPK-PD-L1 binding in (J) and (K).
Hsp90b1, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/hsp90b1/product/Bio-Techne corporation
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
hsp90b1 - by Bioz Stars, 2024-04
86/100 stars

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1) Product Images from "Metformin promotes antitumor immunity via endoplasmic reticulum-associated degradation of PD-L1"

Article Title: Metformin promotes antitumor immunity via endoplasmic reticulum-associated degradation of PD-L1

Journal: Molecular cell

doi: 10.1016/j.molcel.2018.07.030

(A) BT-549 cells were treated with metformin (5 mM) for the indicated time. Detection of endogenous AMPKα and PD-L1 binding (red dots) by Duolink II assay. Three different positions were randomly selected at each point, and the number of red dots were divided by the number of nuclei. Data represent mean ± SD. n = 3. *P, 0.01~0.05, **P, 0.001~0.01, and #P, < 0.001, Student’s t test. Scale bar, 20 μm. Right, MDA-MB-231 WT, PD-L1 KO and AMPKα KO cells were used as negative controls. Scale bar, 25 μm. (B) MDA-MB-231 cells were cultured for 6 hr with or without metformin (5 mM) and MG132 (10 μM). Endogenous PD-L1 and AMPKα were immunoprecipitated and their binding was analyzed with immunoblotting. (C) In vitro kinase activity of AMPK toward PD-L1 with 32P-labeled ATP. (D) Kinetics of PD-L1 phosphorylation by AMPK. Acetyl-CoA carboxylase (ACC) was used as positive control. Km and Vmax were calculated using the Michaelis-Menten equation. (E) In vitro phosphorylation assay and phospho-tag gel shifting assay. W, PD-L1/WT. A, PD-L1/S195A. (F) PD-L1/S195 phosphorylation was examined using anti-PDL1/S195-p antibody at different time points after metformin (5 mM) treatment. (G) Western blot analysis of MDA-MB-231 WT and AMPKα KO cells after metformin treatment (5 mM) for 8 hr. Endogenous PD-L1 purified by IP was subjected to immunoblotting with PDL1 S195-p antibody after PNGase F reaction. (H) PD-L1 and AMPK subcellular localization of MDA-MB-231 WT and AMPKα KO cells. (I) Trypsin digestion of ER fractions with or without permeabilization. (J, K) BT-549 cells were treated with metformin (5 mM) for 3 hr. (J) BT-549 cells were subjected to Duolink II assay combined with immunofluoresence staining using markers for ER (HSP90B1), Golgi (TNG46), and nuclei (Hoechst). Scale bar, 20 μm (inset, 10 μm). (K) Duolink assay with antibodies specific for ECD (Ab205921 and 86744S) and ICD (13684S and GTX104763) of PD-L1. Scale bar, 50 μm (inset, 25 μm). Red dots: AMPK-PD-L1 binding in (J) and (K).
Figure Legend Snippet: (A) BT-549 cells were treated with metformin (5 mM) for the indicated time. Detection of endogenous AMPKα and PD-L1 binding (red dots) by Duolink II assay. Three different positions were randomly selected at each point, and the number of red dots were divided by the number of nuclei. Data represent mean ± SD. n = 3. *P, 0.01~0.05, **P, 0.001~0.01, and #P, < 0.001, Student’s t test. Scale bar, 20 μm. Right, MDA-MB-231 WT, PD-L1 KO and AMPKα KO cells were used as negative controls. Scale bar, 25 μm. (B) MDA-MB-231 cells were cultured for 6 hr with or without metformin (5 mM) and MG132 (10 μM). Endogenous PD-L1 and AMPKα were immunoprecipitated and their binding was analyzed with immunoblotting. (C) In vitro kinase activity of AMPK toward PD-L1 with 32P-labeled ATP. (D) Kinetics of PD-L1 phosphorylation by AMPK. Acetyl-CoA carboxylase (ACC) was used as positive control. Km and Vmax were calculated using the Michaelis-Menten equation. (E) In vitro phosphorylation assay and phospho-tag gel shifting assay. W, PD-L1/WT. A, PD-L1/S195A. (F) PD-L1/S195 phosphorylation was examined using anti-PDL1/S195-p antibody at different time points after metformin (5 mM) treatment. (G) Western blot analysis of MDA-MB-231 WT and AMPKα KO cells after metformin treatment (5 mM) for 8 hr. Endogenous PD-L1 purified by IP was subjected to immunoblotting with PDL1 S195-p antibody after PNGase F reaction. (H) PD-L1 and AMPK subcellular localization of MDA-MB-231 WT and AMPKα KO cells. (I) Trypsin digestion of ER fractions with or without permeabilization. (J, K) BT-549 cells were treated with metformin (5 mM) for 3 hr. (J) BT-549 cells were subjected to Duolink II assay combined with immunofluoresence staining using markers for ER (HSP90B1), Golgi (TNG46), and nuclei (Hoechst). Scale bar, 20 μm (inset, 10 μm). (K) Duolink assay with antibodies specific for ECD (Ab205921 and 86744S) and ICD (13684S and GTX104763) of PD-L1. Scale bar, 50 μm (inset, 25 μm). Red dots: AMPK-PD-L1 binding in (J) and (K).

Techniques Used: Binding Assay, Ii Assay, Cell Culture, Immunoprecipitation, Western Blot, In Vitro, Activity Assay, Labeling, Positive Control, Phosphorylation Assay, Purification, Staining

(A) WT, S195A S195D, S195E, and 4NQ PD-L1 stable cells were treated with or without tunicamycin (5 μg/ml) for 24 hr. (B) Schematic diagram of PD-L1 showing the position of S195 and the 4 N-glycosylation sites. (C) Comparison of the glycan structure between WT and S195E PD-L1 by IP/Mass analysis. (D) BT-549 and MDA-MB-231 stable cells expressing WT, S195E, or S195A PD-L1 were treated with metformin (5 mM) for 24 hr. (E) Expression pattern of PD-L1 in MDA-MB-231 WT, S195A and S195E PD-L1 stable cells by IF staining. (F) MDA-MB-231 stable cells co-stained with antibodies against PD-L1 and Golgi markers (GM130: cis, Giantin: medial, TNG46: trans). (G) IF staining with antibodies against PD-L1 and ER marker (HSP90B1) (H) Flow cytometric analysis of membrane PD-L1 in MDA-MB-231 WT, S195A and S195E PD-L1 stable cells. Data represent mean ± SD. n = 3. (I) Binding of green fluorescent-labeled PD-1/Fc to MDA-MB-231 WT, S195A and S195E PD-L1 stable cells was quantified. Data represent mean ± SD. n = 3. (J) PD-L1 localization in MDA-MB-231 expressing WT, S195E or NXT motif mutant (glycosylation site mutant) PD-L1 by IF staining. For experiments shown in (E), (F), (G) and (J), MG132 (10 μM) was added 6 hr prior to fixation to prevent degradation of PD-L1. Hoechst: nuclear counter staining. Scale bar, 20 μm (inset, 10 μm). *P, 0.01~0.05, **P, 0.001~0.01, and #P, < 0.001, Student’s t test. NS, not significant.
Figure Legend Snippet: (A) WT, S195A S195D, S195E, and 4NQ PD-L1 stable cells were treated with or without tunicamycin (5 μg/ml) for 24 hr. (B) Schematic diagram of PD-L1 showing the position of S195 and the 4 N-glycosylation sites. (C) Comparison of the glycan structure between WT and S195E PD-L1 by IP/Mass analysis. (D) BT-549 and MDA-MB-231 stable cells expressing WT, S195E, or S195A PD-L1 were treated with metformin (5 mM) for 24 hr. (E) Expression pattern of PD-L1 in MDA-MB-231 WT, S195A and S195E PD-L1 stable cells by IF staining. (F) MDA-MB-231 stable cells co-stained with antibodies against PD-L1 and Golgi markers (GM130: cis, Giantin: medial, TNG46: trans). (G) IF staining with antibodies against PD-L1 and ER marker (HSP90B1) (H) Flow cytometric analysis of membrane PD-L1 in MDA-MB-231 WT, S195A and S195E PD-L1 stable cells. Data represent mean ± SD. n = 3. (I) Binding of green fluorescent-labeled PD-1/Fc to MDA-MB-231 WT, S195A and S195E PD-L1 stable cells was quantified. Data represent mean ± SD. n = 3. (J) PD-L1 localization in MDA-MB-231 expressing WT, S195E or NXT motif mutant (glycosylation site mutant) PD-L1 by IF staining. For experiments shown in (E), (F), (G) and (J), MG132 (10 μM) was added 6 hr prior to fixation to prevent degradation of PD-L1. Hoechst: nuclear counter staining. Scale bar, 20 μm (inset, 10 μm). *P, 0.01~0.05, **P, 0.001~0.01, and #P, < 0.001, Student’s t test. NS, not significant.

Techniques Used: Comparison, Expressing, Staining, Marker, Membrane, Binding Assay, Labeling, Mutagenesis


Structured Review

Bio-Techne corporation hsp90b1
(A) BT-549 cells were treated with metformin (5 mM) for the indicated time. Detection of endogenous AMPKα and PD-L1 binding (red dots) by Duolink II assay. Three different positions were randomly selected at each point, and the number of red dots were divided by the number of nuclei. Data represent mean ± SD. n = 3. *P, 0.01~0.05, **P, 0.001~0.01, and #P, < 0.001, Student’s t test. Scale bar, 20 μm. Right, MDA-MB-231 WT, PD-L1 KO and AMPKα KO cells were used as negative controls. Scale bar, 25 μm. (B) MDA-MB-231 cells were cultured for 6 hr with or without metformin (5 mM) and MG132 (10 μM). Endogenous PD-L1 and AMPKα were immunoprecipitated and their binding was analyzed with immunoblotting. (C) In vitro kinase activity of AMPK toward PD-L1 with 32P-labeled ATP. (D) Kinetics of PD-L1 phosphorylation by AMPK. Acetyl-CoA carboxylase (ACC) was used as positive control. Km and Vmax were calculated using the Michaelis-Menten equation. (E) In vitro phosphorylation assay and phospho-tag gel shifting assay. W, PD-L1/WT. A, PD-L1/S195A. (F) PD-L1/S195 phosphorylation was examined using anti-PDL1/S195-p antibody at different time points after metformin (5 mM) treatment. (G) Western blot analysis of MDA-MB-231 WT and AMPKα KO cells after metformin treatment (5 mM) for 8 hr. Endogenous PD-L1 purified by IP was subjected to immunoblotting with PDL1 S195-p antibody after PNGase F reaction. (H) PD-L1 and AMPK subcellular localization of MDA-MB-231 WT and AMPKα KO cells. (I) Trypsin digestion of ER fractions with or without permeabilization. (J, K) BT-549 cells were treated with metformin (5 mM) for 3 hr. (J) BT-549 cells were subjected to Duolink II assay combined with immunofluoresence staining using markers for ER <t>(HSP90B1),</t> Golgi (TNG46), and nuclei (Hoechst). Scale bar, 20 μm (inset, 10 μm). (K) Duolink assay with antibodies specific for ECD (Ab205921 and 86744S) and ICD (13684S and GTX104763) of PD-L1. Scale bar, 50 μm (inset, 25 μm). Red dots: AMPK-PD-L1 binding in (J) and (K).
Hsp90b1, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/hsp90b1/product/Bio-Techne corporation
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
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Images

1) Product Images from "Metformin promotes antitumor immunity via endoplasmic reticulum-associated degradation of PD-L1"

Article Title: Metformin promotes antitumor immunity via endoplasmic reticulum-associated degradation of PD-L1

Journal: Molecular cell

doi: 10.1016/j.molcel.2018.07.030

(A) BT-549 cells were treated with metformin (5 mM) for the indicated time. Detection of endogenous AMPKα and PD-L1 binding (red dots) by Duolink II assay. Three different positions were randomly selected at each point, and the number of red dots were divided by the number of nuclei. Data represent mean ± SD. n = 3. *P, 0.01~0.05, **P, 0.001~0.01, and #P, < 0.001, Student’s t test. Scale bar, 20 μm. Right, MDA-MB-231 WT, PD-L1 KO and AMPKα KO cells were used as negative controls. Scale bar, 25 μm. (B) MDA-MB-231 cells were cultured for 6 hr with or without metformin (5 mM) and MG132 (10 μM). Endogenous PD-L1 and AMPKα were immunoprecipitated and their binding was analyzed with immunoblotting. (C) In vitro kinase activity of AMPK toward PD-L1 with 32P-labeled ATP. (D) Kinetics of PD-L1 phosphorylation by AMPK. Acetyl-CoA carboxylase (ACC) was used as positive control. Km and Vmax were calculated using the Michaelis-Menten equation. (E) In vitro phosphorylation assay and phospho-tag gel shifting assay. W, PD-L1/WT. A, PD-L1/S195A. (F) PD-L1/S195 phosphorylation was examined using anti-PDL1/S195-p antibody at different time points after metformin (5 mM) treatment. (G) Western blot analysis of MDA-MB-231 WT and AMPKα KO cells after metformin treatment (5 mM) for 8 hr. Endogenous PD-L1 purified by IP was subjected to immunoblotting with PDL1 S195-p antibody after PNGase F reaction. (H) PD-L1 and AMPK subcellular localization of MDA-MB-231 WT and AMPKα KO cells. (I) Trypsin digestion of ER fractions with or without permeabilization. (J, K) BT-549 cells were treated with metformin (5 mM) for 3 hr. (J) BT-549 cells were subjected to Duolink II assay combined with immunofluoresence staining using markers for ER (HSP90B1), Golgi (TNG46), and nuclei (Hoechst). Scale bar, 20 μm (inset, 10 μm). (K) Duolink assay with antibodies specific for ECD (Ab205921 and 86744S) and ICD (13684S and GTX104763) of PD-L1. Scale bar, 50 μm (inset, 25 μm). Red dots: AMPK-PD-L1 binding in (J) and (K).
Figure Legend Snippet: (A) BT-549 cells were treated with metformin (5 mM) for the indicated time. Detection of endogenous AMPKα and PD-L1 binding (red dots) by Duolink II assay. Three different positions were randomly selected at each point, and the number of red dots were divided by the number of nuclei. Data represent mean ± SD. n = 3. *P, 0.01~0.05, **P, 0.001~0.01, and #P, < 0.001, Student’s t test. Scale bar, 20 μm. Right, MDA-MB-231 WT, PD-L1 KO and AMPKα KO cells were used as negative controls. Scale bar, 25 μm. (B) MDA-MB-231 cells were cultured for 6 hr with or without metformin (5 mM) and MG132 (10 μM). Endogenous PD-L1 and AMPKα were immunoprecipitated and their binding was analyzed with immunoblotting. (C) In vitro kinase activity of AMPK toward PD-L1 with 32P-labeled ATP. (D) Kinetics of PD-L1 phosphorylation by AMPK. Acetyl-CoA carboxylase (ACC) was used as positive control. Km and Vmax were calculated using the Michaelis-Menten equation. (E) In vitro phosphorylation assay and phospho-tag gel shifting assay. W, PD-L1/WT. A, PD-L1/S195A. (F) PD-L1/S195 phosphorylation was examined using anti-PDL1/S195-p antibody at different time points after metformin (5 mM) treatment. (G) Western blot analysis of MDA-MB-231 WT and AMPKα KO cells after metformin treatment (5 mM) for 8 hr. Endogenous PD-L1 purified by IP was subjected to immunoblotting with PDL1 S195-p antibody after PNGase F reaction. (H) PD-L1 and AMPK subcellular localization of MDA-MB-231 WT and AMPKα KO cells. (I) Trypsin digestion of ER fractions with or without permeabilization. (J, K) BT-549 cells were treated with metformin (5 mM) for 3 hr. (J) BT-549 cells were subjected to Duolink II assay combined with immunofluoresence staining using markers for ER (HSP90B1), Golgi (TNG46), and nuclei (Hoechst). Scale bar, 20 μm (inset, 10 μm). (K) Duolink assay with antibodies specific for ECD (Ab205921 and 86744S) and ICD (13684S and GTX104763) of PD-L1. Scale bar, 50 μm (inset, 25 μm). Red dots: AMPK-PD-L1 binding in (J) and (K).

Techniques Used: Binding Assay, Ii Assay, Cell Culture, Immunoprecipitation, Western Blot, In Vitro, Activity Assay, Labeling, Positive Control, Phosphorylation Assay, Purification, Staining

(A) WT, S195A S195D, S195E, and 4NQ PD-L1 stable cells were treated with or without tunicamycin (5 μg/ml) for 24 hr. (B) Schematic diagram of PD-L1 showing the position of S195 and the 4 N-glycosylation sites. (C) Comparison of the glycan structure between WT and S195E PD-L1 by IP/Mass analysis. (D) BT-549 and MDA-MB-231 stable cells expressing WT, S195E, or S195A PD-L1 were treated with metformin (5 mM) for 24 hr. (E) Expression pattern of PD-L1 in MDA-MB-231 WT, S195A and S195E PD-L1 stable cells by IF staining. (F) MDA-MB-231 stable cells co-stained with antibodies against PD-L1 and Golgi markers (GM130: cis, Giantin: medial, TNG46: trans). (G) IF staining with antibodies against PD-L1 and ER marker (HSP90B1) (H) Flow cytometric analysis of membrane PD-L1 in MDA-MB-231 WT, S195A and S195E PD-L1 stable cells. Data represent mean ± SD. n = 3. (I) Binding of green fluorescent-labeled PD-1/Fc to MDA-MB-231 WT, S195A and S195E PD-L1 stable cells was quantified. Data represent mean ± SD. n = 3. (J) PD-L1 localization in MDA-MB-231 expressing WT, S195E or NXT motif mutant (glycosylation site mutant) PD-L1 by IF staining. For experiments shown in (E), (F), (G) and (J), MG132 (10 μM) was added 6 hr prior to fixation to prevent degradation of PD-L1. Hoechst: nuclear counter staining. Scale bar, 20 μm (inset, 10 μm). *P, 0.01~0.05, **P, 0.001~0.01, and #P, < 0.001, Student’s t test. NS, not significant.
Figure Legend Snippet: (A) WT, S195A S195D, S195E, and 4NQ PD-L1 stable cells were treated with or without tunicamycin (5 μg/ml) for 24 hr. (B) Schematic diagram of PD-L1 showing the position of S195 and the 4 N-glycosylation sites. (C) Comparison of the glycan structure between WT and S195E PD-L1 by IP/Mass analysis. (D) BT-549 and MDA-MB-231 stable cells expressing WT, S195E, or S195A PD-L1 were treated with metformin (5 mM) for 24 hr. (E) Expression pattern of PD-L1 in MDA-MB-231 WT, S195A and S195E PD-L1 stable cells by IF staining. (F) MDA-MB-231 stable cells co-stained with antibodies against PD-L1 and Golgi markers (GM130: cis, Giantin: medial, TNG46: trans). (G) IF staining with antibodies against PD-L1 and ER marker (HSP90B1) (H) Flow cytometric analysis of membrane PD-L1 in MDA-MB-231 WT, S195A and S195E PD-L1 stable cells. Data represent mean ± SD. n = 3. (I) Binding of green fluorescent-labeled PD-1/Fc to MDA-MB-231 WT, S195A and S195E PD-L1 stable cells was quantified. Data represent mean ± SD. n = 3. (J) PD-L1 localization in MDA-MB-231 expressing WT, S195E or NXT motif mutant (glycosylation site mutant) PD-L1 by IF staining. For experiments shown in (E), (F), (G) and (J), MG132 (10 μM) was added 6 hr prior to fixation to prevent degradation of PD-L1. Hoechst: nuclear counter staining. Scale bar, 20 μm (inset, 10 μm). *P, 0.01~0.05, **P, 0.001~0.01, and #P, < 0.001, Student’s t test. NS, not significant.

Techniques Used: Comparison, Expressing, Staining, Marker, Membrane, Binding Assay, Labeling, Mutagenesis


Structured Review

Bio-Techne corporation hsp90b1
(A) IP followed by WB analysis of JAK1, STT3A, and ngPD-L1 tyrosine phosphorylation (4G10) in FLAG–ngPD-L1–SK-HEP-1 cells with or without exposure to IL-6 (20 ng/mL) and ruxolitinib (10 μmol/L) for 30 minutes. (B) JAK1 interacts with ngPD-L1 in ER lumen. Representative images of individual immunofluorescence staining of JAK1 and PD-L1 interaction in ER region in Hep 3B cells by Duolink assay. The red dots (JAK1/PD-L1 interaction) indicate their interaction. Green fluorescence <t>(HSP90B1)</t> was used as ER marker, and DAPI as a nuclear marker. (C) Schematic showing JAK1/PD-L1 interaction in the ER. IC, intracellular domain; TM, transmembrane domain; EC, extracellular domain. (D) Trypsin digestion of ER fractions with (group 3) or without (group 2) permeabilization in Hep 3B cells.
Hsp90b1, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/hsp90b1/product/Bio-Techne corporation
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
hsp90b1 - by Bioz Stars, 2024-04
86/100 stars

Images

1) Product Images from "IL-6/JAK1 pathway drives PD-L1 Y112 phosphorylation to promote cancer immune evasion"

Article Title: IL-6/JAK1 pathway drives PD-L1 Y112 phosphorylation to promote cancer immune evasion

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI126022

(A) IP followed by WB analysis of JAK1, STT3A, and ngPD-L1 tyrosine phosphorylation (4G10) in FLAG–ngPD-L1–SK-HEP-1 cells with or without exposure to IL-6 (20 ng/mL) and ruxolitinib (10 μmol/L) for 30 minutes. (B) JAK1 interacts with ngPD-L1 in ER lumen. Representative images of individual immunofluorescence staining of JAK1 and PD-L1 interaction in ER region in Hep 3B cells by Duolink assay. The red dots (JAK1/PD-L1 interaction) indicate their interaction. Green fluorescence (HSP90B1) was used as ER marker, and DAPI as a nuclear marker. (C) Schematic showing JAK1/PD-L1 interaction in the ER. IC, intracellular domain; TM, transmembrane domain; EC, extracellular domain. (D) Trypsin digestion of ER fractions with (group 3) or without (group 2) permeabilization in Hep 3B cells.
Figure Legend Snippet: (A) IP followed by WB analysis of JAK1, STT3A, and ngPD-L1 tyrosine phosphorylation (4G10) in FLAG–ngPD-L1–SK-HEP-1 cells with or without exposure to IL-6 (20 ng/mL) and ruxolitinib (10 μmol/L) for 30 minutes. (B) JAK1 interacts with ngPD-L1 in ER lumen. Representative images of individual immunofluorescence staining of JAK1 and PD-L1 interaction in ER region in Hep 3B cells by Duolink assay. The red dots (JAK1/PD-L1 interaction) indicate their interaction. Green fluorescence (HSP90B1) was used as ER marker, and DAPI as a nuclear marker. (C) Schematic showing JAK1/PD-L1 interaction in the ER. IC, intracellular domain; TM, transmembrane domain; EC, extracellular domain. (D) Trypsin digestion of ER fractions with (group 3) or without (group 2) permeabilization in Hep 3B cells.

Techniques Used: Immunofluorescence, Staining, Fluorescence, Marker

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    Bio-Techne corporation gp96
    a , Relative expression of ETV3 or ETV6 in blood monocytes, mo-Macs and mo-DCs isolated from peritoneal ascites or generated in vitro (accession nos. GSE102046 and GSE40484 ). a.u., arbitrary units. b – g , Monocytes were cultured with M-CSF, IL-4 and TNF. b , At day 5, mo-Macs and mo-DCs were sorted and lysed for immunoblot analysis. <t>GP96</t> was used as a loading control. Representative results are shown ( n = 5), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). c – f , ETV3 or ETV6 expression was silenced using a lentivirus-containing shRNA. c , e , Protein quantification by immunoblotting after 5 d for ETV3 (c) or ETV6 (e). Actin was used as a loading control. Representative results are shown ( n = 8), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). d , f , Mo-mac and mo-DC differentiation from monocytes after 5 d of ETV3 (d) or ETV6 (f) silencing . One representative donor is shown ( n = 8) and the median ( n = 8 in three independent experiments; paired one-way analysis of variance (ANOVA)). DN, double negative. g , ETV3 or ETV6 mRNA expression was analyzed by RT–qPCR. Each symbol represents an individual donor ( n = 6 in three independent experiments). h , Monocytes were cultured for 3 and 6 h with medium only or combinations of M-CSF, IL-4 and TNF. Each symbol represents an individual donor ( n = 5 in two independent experiments; paired one-way ANOVA). For all panels: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All statistical tests were two sided. For immunoblots, paired samples were derived from the same experiment and processed in parallel.
    Gp96, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a , Relative expression of ETV3 or ETV6 in blood monocytes, mo-Macs and mo-DCs isolated from peritoneal ascites or generated in vitro (accession nos. GSE102046 and GSE40484 ). a.u., arbitrary units. b – g , Monocytes were cultured with M-CSF, IL-4 and TNF. b , At day 5, mo-Macs and mo-DCs were sorted and lysed for immunoblot analysis. <t>GP96</t> was used as a loading control. Representative results are shown ( n = 5), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). c – f , ETV3 or ETV6 expression was silenced using a lentivirus-containing shRNA. c , e , Protein quantification by immunoblotting after 5 d for ETV3 (c) or ETV6 (e). Actin was used as a loading control. Representative results are shown ( n = 8), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). d , f , Mo-mac and mo-DC differentiation from monocytes after 5 d of ETV3 (d) or ETV6 (f) silencing . One representative donor is shown ( n = 8) and the median ( n = 8 in three independent experiments; paired one-way analysis of variance (ANOVA)). DN, double negative. g , ETV3 or ETV6 mRNA expression was analyzed by RT–qPCR. Each symbol represents an individual donor ( n = 6 in three independent experiments). h , Monocytes were cultured for 3 and 6 h with medium only or combinations of M-CSF, IL-4 and TNF. Each symbol represents an individual donor ( n = 5 in two independent experiments; paired one-way ANOVA). For all panels: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All statistical tests were two sided. For immunoblots, paired samples were derived from the same experiment and processed in parallel.
    Hsp90b1, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hsp90b1/product/Bio-Techne corporation
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    a , Relative expression of ETV3 or ETV6 in blood monocytes, mo-Macs and mo-DCs isolated from peritoneal ascites or generated in vitro (accession nos. GSE102046 and GSE40484 ). a.u., arbitrary units. b – g , Monocytes were cultured with M-CSF, IL-4 and TNF. b , At day 5, mo-Macs and mo-DCs were sorted and lysed for immunoblot analysis. <t>GP96</t> was used as a loading control. Representative results are shown ( n = 5), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). c – f , ETV3 or ETV6 expression was silenced using a lentivirus-containing shRNA. c , e , Protein quantification by immunoblotting after 5 d for ETV3 (c) or ETV6 (e). Actin was used as a loading control. Representative results are shown ( n = 8), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). d , f , Mo-mac and mo-DC differentiation from monocytes after 5 d of ETV3 (d) or ETV6 (f) silencing . One representative donor is shown ( n = 8) and the median ( n = 8 in three independent experiments; paired one-way analysis of variance (ANOVA)). DN, double negative. g , ETV3 or ETV6 mRNA expression was analyzed by RT–qPCR. Each symbol represents an individual donor ( n = 6 in three independent experiments). h , Monocytes were cultured for 3 and 6 h with medium only or combinations of M-CSF, IL-4 and TNF. Each symbol represents an individual donor ( n = 5 in two independent experiments; paired one-way ANOVA). For all panels: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All statistical tests were two sided. For immunoblots, paired samples were derived from the same experiment and processed in parallel.
    Anti Hsp90b1, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Techne corporation anti mouse gp96
    Expression of gB and <t>GP96‐NT</t> plasmids in vitro and in vivo. 3T3 cells were transfected with pgB, pGP96‐NT or vector with PEI for 48 h, and then, cell lysates were subjected to Western blot analysis using anti‐gB, anti‐GP96 or anti‐β‐actin antibody, respectively. A, In vitro expression of gB and GP96‐NT plasmids. Lane 1: 3T3 cells transfected with pcDNA3.1‐GP96‐NT; lane 2: 3T3 cells transfected with pcDNA3.1 vector; lane 3: 3T3 cells transfected with pcDNA3.1‐gB; lane 4: 3T3 cells transfected with pcDNA3.1 vector. B, Quantification of gB and GP96‐NT expression by densitometry in vitro. Data were from one representative experiment of 3 performed and presented as the mean ± SD, and t test was used, *** P < .001. C, Balb/c mice were intranasally immunized with PEI‐pgB or PEI‐pGP96‐NT vaccines, respectively, and lungs were taken 3 days later for gene expression analysis. Western blot analysis of gB, GP96 or β‐actin expression for lung tissues from immunized mice. Lane 1: Mice immunized with pcDNA3.1‐GP96‐NT; lane 2: mice immunized with pcDNA3.1 vector; lane 3: mice immunized with pcDNA3.1‐gB; lane 4: mice immunized with pcDNA3.1 vector. D, Quantification of gB and GP96‐NT expression by densitometry in vivo. Data were from one representative experiment of 3 performed and presented as the mean ± SD (n = 8), and t test was used,*** P < .001
    Anti Mouse Gp96, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Techne corporation 143 hsp90b1
    Expression of gB and <t>GP96‐NT</t> plasmids in vitro and in vivo. 3T3 cells were transfected with pgB, pGP96‐NT or vector with PEI for 48 h, and then, cell lysates were subjected to Western blot analysis using anti‐gB, anti‐GP96 or anti‐β‐actin antibody, respectively. A, In vitro expression of gB and GP96‐NT plasmids. Lane 1: 3T3 cells transfected with pcDNA3.1‐GP96‐NT; lane 2: 3T3 cells transfected with pcDNA3.1 vector; lane 3: 3T3 cells transfected with pcDNA3.1‐gB; lane 4: 3T3 cells transfected with pcDNA3.1 vector. B, Quantification of gB and GP96‐NT expression by densitometry in vitro. Data were from one representative experiment of 3 performed and presented as the mean ± SD, and t test was used, *** P < .001. C, Balb/c mice were intranasally immunized with PEI‐pgB or PEI‐pGP96‐NT vaccines, respectively, and lungs were taken 3 days later for gene expression analysis. Western blot analysis of gB, GP96 or β‐actin expression for lung tissues from immunized mice. Lane 1: Mice immunized with pcDNA3.1‐GP96‐NT; lane 2: mice immunized with pcDNA3.1 vector; lane 3: mice immunized with pcDNA3.1‐gB; lane 4: mice immunized with pcDNA3.1 vector. D, Quantification of gB and GP96‐NT expression by densitometry in vivo. Data were from one representative experiment of 3 performed and presented as the mean ± SD (n = 8), and t test was used,*** P < .001
    143 Hsp90b1, supplied by Bio-Techne corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/143 hsp90b1/product/Bio-Techne corporation
    Average 86 stars, based on 1 article reviews
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    143 hsp90b1 - by Bioz Stars, 2024-04
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    Image Search Results


    a , Relative expression of ETV3 or ETV6 in blood monocytes, mo-Macs and mo-DCs isolated from peritoneal ascites or generated in vitro (accession nos. GSE102046 and GSE40484 ). a.u., arbitrary units. b – g , Monocytes were cultured with M-CSF, IL-4 and TNF. b , At day 5, mo-Macs and mo-DCs were sorted and lysed for immunoblot analysis. GP96 was used as a loading control. Representative results are shown ( n = 5), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). c – f , ETV3 or ETV6 expression was silenced using a lentivirus-containing shRNA. c , e , Protein quantification by immunoblotting after 5 d for ETV3 (c) or ETV6 (e). Actin was used as a loading control. Representative results are shown ( n = 8), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). d , f , Mo-mac and mo-DC differentiation from monocytes after 5 d of ETV3 (d) or ETV6 (f) silencing . One representative donor is shown ( n = 8) and the median ( n = 8 in three independent experiments; paired one-way analysis of variance (ANOVA)). DN, double negative. g , ETV3 or ETV6 mRNA expression was analyzed by RT–qPCR. Each symbol represents an individual donor ( n = 6 in three independent experiments). h , Monocytes were cultured for 3 and 6 h with medium only or combinations of M-CSF, IL-4 and TNF. Each symbol represents an individual donor ( n = 5 in two independent experiments; paired one-way ANOVA). For all panels: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All statistical tests were two sided. For immunoblots, paired samples were derived from the same experiment and processed in parallel.

    Journal: Nature Immunology

    Article Title: ETV3 and ETV6 enable monocyte differentiation into dendritic cells by repressing macrophage fate commitment

    doi: 10.1038/s41590-022-01374-0

    Figure Lengend Snippet: a , Relative expression of ETV3 or ETV6 in blood monocytes, mo-Macs and mo-DCs isolated from peritoneal ascites or generated in vitro (accession nos. GSE102046 and GSE40484 ). a.u., arbitrary units. b – g , Monocytes were cultured with M-CSF, IL-4 and TNF. b , At day 5, mo-Macs and mo-DCs were sorted and lysed for immunoblot analysis. GP96 was used as a loading control. Representative results are shown ( n = 5), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). c – f , ETV3 or ETV6 expression was silenced using a lentivirus-containing shRNA. c , e , Protein quantification by immunoblotting after 5 d for ETV3 (c) or ETV6 (e). Actin was used as a loading control. Representative results are shown ( n = 8), quantification was performed by densitometry and each symbol represents an individual donor (paired Student’s t -test). d , f , Mo-mac and mo-DC differentiation from monocytes after 5 d of ETV3 (d) or ETV6 (f) silencing . One representative donor is shown ( n = 8) and the median ( n = 8 in three independent experiments; paired one-way analysis of variance (ANOVA)). DN, double negative. g , ETV3 or ETV6 mRNA expression was analyzed by RT–qPCR. Each symbol represents an individual donor ( n = 6 in three independent experiments). h , Monocytes were cultured for 3 and 6 h with medium only or combinations of M-CSF, IL-4 and TNF. Each symbol represents an individual donor ( n = 5 in two independent experiments; paired one-way ANOVA). For all panels: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All statistical tests were two sided. For immunoblots, paired samples were derived from the same experiment and processed in parallel.

    Article Snippet: Membranes were stained with primary antibodies against ETV6/Tel (Novus Biologicals, catalog no. NBP1-80695, 0.4 μg ml −1 ), ETV3 (Atlas Antibodies, catalog no. HPA004794, 0.4 μg ml −1 ), GP96 (Novus Biologicals, clone 9G10, 0.4 μg ml −1 ) or actin (Millipore, clone C4, 0.4 μg ml −1 ), followed by horeseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch, dilution 1:10,000).

    Techniques: Expressing, Isolation, Generated, In Vitro, Cell Culture, Western Blot, shRNA, Quantitative RT-PCR, Derivative Assay

    a – d , f , g , Monocytes were cultured with M-CSF, IL-4 and TNF for 3 d. ChIP–seq analysis was performed for ETV3 or ETV6. a , Motif enrichment analysis obtained with HOMER. b , Most enriched motifs. c , Overlap of identified genes for ETV3 or ETV6 immunoprecipitation (IP). Peaks in all gene regions or only in the transcription start site (TSS) region are shown. d , Overlap of DEGs found in RNA-seq and genes identified in ChIP–seq. e , f , Data from RNA-seq analysis. e , GSEA of monocyte, mo-Mac and mo-DC signatures in control (red) versus silenced (blue) samples. NES, normalized enrichment score. f , Heatmap of DEGs belonging to the monocyte, mo-Mac or mo-DC signatures. Samples were ordered by condition and genes were ordered manually. Genes detected in ChIP–seq analysis are emboldened. g , Gene tracks from ChIP–seq analysis for the genomic region of MAFB . h – j , Monocytes were cultured with M-CSF for 5 d. ETV6 was overexpressed using a lentivirus containing an expression plasmid. h , Protein quantification by immunblotting. GP96 was used as a loading control. Representative results are shown ( n = 5 in two independent experiments). Paired samples derive from the same experiment and were processed in parallel. i , Mo-Mac differentiation. One representative donor is shown ( n = 11). j , Percentage of mo-Macs after 5 d ( n = 11 in four independent experiments; paired Student’s t -test: *** P < 0.001). All statistical tests were two sided.

    Journal: Nature Immunology

    Article Title: ETV3 and ETV6 enable monocyte differentiation into dendritic cells by repressing macrophage fate commitment

    doi: 10.1038/s41590-022-01374-0

    Figure Lengend Snippet: a – d , f , g , Monocytes were cultured with M-CSF, IL-4 and TNF for 3 d. ChIP–seq analysis was performed for ETV3 or ETV6. a , Motif enrichment analysis obtained with HOMER. b , Most enriched motifs. c , Overlap of identified genes for ETV3 or ETV6 immunoprecipitation (IP). Peaks in all gene regions or only in the transcription start site (TSS) region are shown. d , Overlap of DEGs found in RNA-seq and genes identified in ChIP–seq. e , f , Data from RNA-seq analysis. e , GSEA of monocyte, mo-Mac and mo-DC signatures in control (red) versus silenced (blue) samples. NES, normalized enrichment score. f , Heatmap of DEGs belonging to the monocyte, mo-Mac or mo-DC signatures. Samples were ordered by condition and genes were ordered manually. Genes detected in ChIP–seq analysis are emboldened. g , Gene tracks from ChIP–seq analysis for the genomic region of MAFB . h – j , Monocytes were cultured with M-CSF for 5 d. ETV6 was overexpressed using a lentivirus containing an expression plasmid. h , Protein quantification by immunblotting. GP96 was used as a loading control. Representative results are shown ( n = 5 in two independent experiments). Paired samples derive from the same experiment and were processed in parallel. i , Mo-Mac differentiation. One representative donor is shown ( n = 11). j , Percentage of mo-Macs after 5 d ( n = 11 in four independent experiments; paired Student’s t -test: *** P < 0.001). All statistical tests were two sided.

    Article Snippet: Membranes were stained with primary antibodies against ETV6/Tel (Novus Biologicals, catalog no. NBP1-80695, 0.4 μg ml −1 ), ETV3 (Atlas Antibodies, catalog no. HPA004794, 0.4 μg ml −1 ), GP96 (Novus Biologicals, clone 9G10, 0.4 μg ml −1 ) or actin (Millipore, clone C4, 0.4 μg ml −1 ), followed by horeseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch, dilution 1:10,000).

    Techniques: Cell Culture, ChIP-sequencing, Immunoprecipitation, RNA Sequencing Assay, Expressing, Plasmid Preparation

    Expression of gB and GP96‐NT plasmids in vitro and in vivo. 3T3 cells were transfected with pgB, pGP96‐NT or vector with PEI for 48 h, and then, cell lysates were subjected to Western blot analysis using anti‐gB, anti‐GP96 or anti‐β‐actin antibody, respectively. A, In vitro expression of gB and GP96‐NT plasmids. Lane 1: 3T3 cells transfected with pcDNA3.1‐GP96‐NT; lane 2: 3T3 cells transfected with pcDNA3.1 vector; lane 3: 3T3 cells transfected with pcDNA3.1‐gB; lane 4: 3T3 cells transfected with pcDNA3.1 vector. B, Quantification of gB and GP96‐NT expression by densitometry in vitro. Data were from one representative experiment of 3 performed and presented as the mean ± SD, and t test was used, *** P < .001. C, Balb/c mice were intranasally immunized with PEI‐pgB or PEI‐pGP96‐NT vaccines, respectively, and lungs were taken 3 days later for gene expression analysis. Western blot analysis of gB, GP96 or β‐actin expression for lung tissues from immunized mice. Lane 1: Mice immunized with pcDNA3.1‐GP96‐NT; lane 2: mice immunized with pcDNA3.1 vector; lane 3: mice immunized with pcDNA3.1‐gB; lane 4: mice immunized with pcDNA3.1 vector. D, Quantification of gB and GP96‐NT expression by densitometry in vivo. Data were from one representative experiment of 3 performed and presented as the mean ± SD (n = 8), and t test was used,*** P < .001

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: gB co‐immunization with GP96 enhances pulmonary‐resident CD8 T cells and exerts a long‐term defence against MCMV pneumonitis

    doi: 10.1111/jcmm.16065

    Figure Lengend Snippet: Expression of gB and GP96‐NT plasmids in vitro and in vivo. 3T3 cells were transfected with pgB, pGP96‐NT or vector with PEI for 48 h, and then, cell lysates were subjected to Western blot analysis using anti‐gB, anti‐GP96 or anti‐β‐actin antibody, respectively. A, In vitro expression of gB and GP96‐NT plasmids. Lane 1: 3T3 cells transfected with pcDNA3.1‐GP96‐NT; lane 2: 3T3 cells transfected with pcDNA3.1 vector; lane 3: 3T3 cells transfected with pcDNA3.1‐gB; lane 4: 3T3 cells transfected with pcDNA3.1 vector. B, Quantification of gB and GP96‐NT expression by densitometry in vitro. Data were from one representative experiment of 3 performed and presented as the mean ± SD, and t test was used, *** P < .001. C, Balb/c mice were intranasally immunized with PEI‐pgB or PEI‐pGP96‐NT vaccines, respectively, and lungs were taken 3 days later for gene expression analysis. Western blot analysis of gB, GP96 or β‐actin expression for lung tissues from immunized mice. Lane 1: Mice immunized with pcDNA3.1‐GP96‐NT; lane 2: mice immunized with pcDNA3.1 vector; lane 3: mice immunized with pcDNA3.1‐gB; lane 4: mice immunized with pcDNA3.1 vector. D, Quantification of gB and GP96‐NT expression by densitometry in vivo. Data were from one representative experiment of 3 performed and presented as the mean ± SD (n = 8), and t test was used,*** P < .001

    Article Snippet: The membrane was probed with anti‐mouse GP96 (Novus Biologicals, dilution 1:1000), mouse anti‐gB antibody (Capri, dilution 1:1000) or anti‐β‐actin (CST, dilution 1:1000) followed by goat anti‐mouse/rabbit IgG‐HRP (Southern Biotech, dilution 1:5000).

    Techniques: Expressing, In Vitro, In Vivo, Transfection, Plasmid Preparation, Western Blot, Vaccines