gp96 (Oxford Instruments)
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Oxford Instruments
gp96
Gp96, supplied by Oxford Instruments, used in various techniques. Bioz Stars score: 99/100, based on 41025 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gp96/product/Oxford Instruments
Average 99 stars, based on 41025 article reviews
Gp96, supplied by Oxford Instruments, used in various techniques. Bioz Stars score: 99/100, based on 41025 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gp96/product/Oxford Instruments
Average 99 stars, based on 41025 article reviews
gp96 - by Bioz Stars,
2026-03
99/100 stars
Images
1) Product Images from "Heat shock protein gp96 drives natural killer cell maturation and anti-tumor immunity by counteracting Trim28 to stabilize Eomes"
Article Title: Heat shock protein gp96 drives natural killer cell maturation and anti-tumor immunity by counteracting Trim28 to stabilize Eomes
Journal: Nature Communications
doi: 10.1038/s41467-024-45426-5
Figure Legend Snippet: A Western blot of gp96 expression in splenic NK cells sorted from WT and KO mice. Representative flow cytometry plots showing the percentages of NKp (NK1.1 - DX5 - ), iNK (NK1.1 + DX5 - ), mNK (NK1.1 + DX5 + ) cells gated on CD3 - CD122 + splenocytes ( B ) and bone marrow ( C ) cells and representative flow cytometry plots showing the percentages of CD27 - CD11b - (DN), CD27 + CD11b - (CD27 SP), CD27 + CD11b + (DP), and CD27 - CD11b + (CD11b SP) cells on gated NK1.1 + DX5 + splenocytes ( B ) and bone marrow ( C ) cells from WT and gp96-deficient mice. The numbers are percentages of the indicated quadrants among the gated cells. D Flow cytometry analysis of indicated marker levels. E Expression of indicated markers as determined by RNA-seq. The fold change indicates the difference in relative transcript expression between WT compared with Ncr1 Cre gp96 fl/fl mice. F – H The chimeric mouse model was produced as in ( F ). Flow cytometry analysis of CD27 and CD11b on DX5 + NK cells from CD45.1 + cells and CD45.2 + cells in the spleen ( G ) and bone marrow ( H ). The data are representative of two independent experiments with similar results. Dots represent data from n = 5 mice/group. Mean ± SD is shown. Statistical significance was determined using two-tailed unpaired t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. p values: ( B ) p = 0.002 (NKp), p < 0.0001 (iNK), p < 0.0001 (mNK), p < 0.0001 (DN), p < 0.0001 (CD27 SP), p < 0.0001 (DP), p < 0.0001 (CD11b SP), ( C ) p = 0.009 (NKp), p < 0.0001 (iNK), p < 0.0001 (mNK), p = 0.0427 (DN), p < 0.0001 (CD27 SP), p < 0.0001 (DP), p < 0.0001 (CD11b SP), ( G ) p < 0.0001 (NKp), p < 0.0001 (iNK), p < 0.0001 (mNK), p < 0.0001 (DN), p = 0.0011 (CD27 SP), p = 0.0002 (DP), p < 0.0001 (CD11b SP), ( H ) p = 0.0281 (NKp), p < 0.0001 (iNK), p < 0.0001 (mNK), p < 0.0001 (DN), p = 0.0002 (CD27 SP), p = 0.0008 (DP), p < 0.0001 (CD11b SP).
Techniques Used: Western Blot, Expressing, Flow Cytometry, Marker, RNA Sequencing, Produced, Two Tailed Test
Figure Legend Snippet: A A t-Distributed Stochastic Neighbor Embedding (tSNE) and graph visualization of the 15699 single NK cells defining 6 clusters. B Heatmap of marker genes in scRNA-seq clusters. Columns: single cells. Rows: cluster marker genes. Representative genes that are differentially expressed are on the left. C Cell number of Ncr1 Cre gp96 fl/fl and WT NK cells within each cluster. D Feature dot plot showing the relative expression levels of the indicated genes in each cluster from ( A ). E Velocity analysis of the origin and direction of NK cell maturation. Velocity fields were projected onto the t-SNE plot. F KEGG analysis of DEGs for indicated clusters. Statistical significance was determined using one-sided Fisher’s Exact Test with adjustments for multiple comparisons typically using methods like the Benjamini-Hochberg procedure. Selected KEGG terms with adjusted P values < 0.05 are shown.
Techniques Used: Marker, Expressing
Figure Legend Snippet: A The fold changes indicated the difference in relative transcript expression of Eomes-bound genes in DX5 + splenic NK cells between WT compared with Ncr1 Cre gp96 fl/fl mice, as determined by RNA-seq. Y-axis showed Log2 fold changes of WT vs. KO mice. B Correlation analysis of Log 2 fold changes of Eomes-targeted genes between gp96 –/– NK versus WT NK and Eomes –/– versus WT NK. C Flow cytometry analysis of levels of Eomes between Ncr1 Cre gp96 fl/fl and WT NK cells. D Western blot analysis of Eomes levels between Ncr1 Cre gp96 fl/fl and WT NK cells from spleen and bone marrow (BM). E Flow cytometry analysis of DX5 + NK cell percentage in WT or gp96 KO BM hematopoietic stem cells infected with Eomes or control Lentiviral vector. Mean ± SD of three replicates is shown. Statistical significance was determined using two-tailed unpaired t test. * p < 0.05, *** p < 0.001, **** p < 0.0001. p values: ( B ) p = 0.0004, ( C ) p < 0.0001 (spleen), p = 0.0002 (BM), ( D ) p < 0.0001 (spleen), p < 0.0001 (BM), ( E ) p = 0.0003 (WT-control vs KO-control), p = 0.0117 (KO-control vs KO-Eomes).
Techniques Used: Expressing, RNA Sequencing, Flow Cytometry, Western Blot, Infection, Control, Plasmid Preparation, Two Tailed Test
Figure Legend Snippet: A Flow cytometry analysis of Eomes and gp96 levels among CD27 single positive, double positive (DP), CD11b single positive NK cells in mouse spleen. Dots represent data from n = 5 mice/group. B Immunostaining of gp96 in spleen NK cells sorted from WT mice. The mean fluorescence intensity of gp96 was analyzed by Imaris 9.7. Bar, 10 μm. C – E CD11b + CD27 + double positive NK cells were sorted from WT mice and subsequently stained for nucleus (Hoechst), Eomes, gp96, Calregulin and α-tubulin for confocal microscopy analysis. Scale bars, 3 μm for original shots ( C – E ) and 1 μm for magnified views ( C ). Z-stack images of gp96 and Eomes in whole cell, cytosol and nucleus, respectively. Percentage of ROI colocalized for cytosol and nucleus were obtained by Imaris. Percentages of colocalization of gp96 and Trim28 in the nucleus and cytosol were calculated, respectively. Scale bars, 3 μm. Dots represent data from n = 3 fields. ( D ). F Western blot analysis of Eomes levels in spleen NK cells sorted from WT and gp96 KO mice. Cells were treated with 50 μg/ml CHX for the indicated times. The band intensity at 0 h in WT NK cells was arbitrarily taken as 1.0. G Western blot analysis of Eomes levels in 293 cells transfected with Flag-gp96 or the empty vector. Cells were treated with 50 μg/ml CHX for the indicated times. The band intensity at 0 h in vector cells was arbitrarily taken as 1.0. H 293 cells stably expressing Eomes (left) and NK cells from gp96 KO mice (right) were treated with either 40 µM CQ or 10 µM MG132 for 6 h and subjected to western blotting. I WT and Atg5 knockout 293 cells were transfected with His-Eomes and Flag-gp96 or a control vector. Cells were lysed and subjected to western blotting. J WT, Atg5 and gp96/Atg5 double knockout 293 cells were transfected with His-Eomes and subjected to western blotting. K WT and gp96 knockout HEK293 cells were transfected with His-Eomes. Cells were grown for 24 h and treated with 20 nM of BafA1 for 4 h, followed by IP-Western analyses. L WT and gp96 knockout 293 cells were co-transfected with His-Eomes and GFP-P62. Cells were then treated as ( H ) and subjected to IP-Western analyses. The data are representative of two independent experiments with similar results. Mean ± SD is shown. Statistical significance was determined using two-tailed unpaired t test. * p < 0.05, ** p < 0.01, **** p < 0.0001. p values: (A) p < 0.0001 (Eomes, CD27 SP vs DP), p < 0.0001 (Eomes, DP vs CD11b SP), p = 0.0193 (gp96, CD27 SP vs DP), p = 0.0391 (gp96, DP vs CD11b SP), ( B ) p < 0.0001 (CD27 SP vs DP), p = 0.0097 (DP vs CD11b SP).
Techniques Used: Flow Cytometry, Immunostaining, Fluorescence, Staining, Confocal Microscopy, Western Blot, Transfection, Plasmid Preparation, Stable Transfection, Expressing, Knock-Out, Control, Double Knockout, Two Tailed Test
Figure Legend Snippet: A , B HEK293 cells stably expressing Eomes were transfected with siRNAs targeting indicated E3 ligases. Cells were then subjected to western blotting ( A ). Cells were grown for 24 h and treated with 20 nM of BafA1 for 4 h, followed by IP-Western analyses ( B ). C HEK293 cells stably expressing Eomes were transfected with Trim28 or a control vector. Cells were treated with 50 μg/ml CHX for the time as indicated and were then subjected to western blotting. D Flow cytometry analysis of Eomes levels in GFP - and GFP + cell in primary NK cells infected with Trim28 GFP lentiviral vector (MOI = 10). n = 3 biologically independent samples. Mean ± SD is shown. Primary NK cells transfected with Trim28 were treated with 20 nM of BafA1 for 4 h, followed by IP-Western analyses ( E ), or endogenous Eomes protein was immunoprecipitated with a specific antibody for Eomes or normal rabbit IgG, followed by immunoblotting ( F ). G The protein-protein interaction prediction tools-ZDOCK 3.0.2 was used to predict the interaction between full-length Eomes (PDB ID: AF-O54839) and Trim28 (PDB ID: AF-Q62318). Blue represents Trim28, and the green represents Eomes. H HEK293 cells transfected with indicated plasmids were grown for 24 h and treated with 20 nM of BafA1 for 4 h, followed by IP-Western analyses. I WT and gp96 knockout HEK293 cells were transfected with His-Eomes and HA-Trim28 plasmids. Cells were grown for 24 h and treated with 20 nM of BafA1 for 4 h, followed by IP-Western analyses. J HEK293 cells stably expressing Eomes were transfected with indicated plasmids. Cells were grown for 24 h, followed by IP-Western analyses. K WT and gp96 knockout HEK293 cells were transfected with His-Eomes and HA-Trim28 plasmids. Cells were grown for 24 h, followed by IP-Western analyses. L Primary NK cells from WT and gp96 knockout mice were sorted, and endogenous Eomes protein was immunoprecipitated with a specific antibody for Eomes or normal rabbit IgG, followed by immunoblotting. M HEK293 cells stably expressing Flag-gp96 were transfected with indicated plasmids. Cells were grown for 24 h, followed by IP-Western analyses. N The protein-protein interaction prediction tools-ZDOCK 3.0.2 were used to predict the interaction between Eomes (PDB ID: AF-O54839) and Trim28 (PDB ID: AF-Q62318). Blue represents the Ring domain of Trim28, and the green represents Eomes. O The protein-protein interaction prediction tools-ZDOCK 3.0.2 were used to predict the interaction between gp96 (PDB ID: AF-P14625) and Trim28 (PDB ID: AF-Q62318). Blue represents the Ring domain of Trim28, and the green represents gp96. P A working model. The E3 ubiquitin ligase Trim28 targets Eomes for lysosomal degradation, resulting in the inhibition of NK development and function. Gp96 binds to Trim28 mainly in cytosol and Eomes mainly in nucleus, and protects Eomes from Trim28-mediated degradation. The data are representative of two independent experiments with similar results.
Techniques Used: Stable Transfection, Expressing, Transfection, Western Blot, Control, Plasmid Preparation, Flow Cytometry, Infection, Immunoprecipitation, Knock-Out, Ubiquitin Proteomics, Inhibition
![CCDC134 controls <t>Gp96</t> protein glycosylation and stability. (A) Immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. (B) Volcano plot of proteins, identified by mass spectrometry, from Flag-immunoprecipitates from sg CCDC134 CAL-1 stably reconstituted with Flag-CCDC134 or control CAL-1 (sg Ren transduced with empty vector [EV]). red: fold change (FC) >2; gray: fold change (FC) = [−2, 2]; blue: fold change (FC) less than −2. (C) Immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. (D) Coomassie blue staining of recombinant proteins Gp96-HA or Flag-CCDC134 (Flag-134) in a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Red arrow indicates specific Gp96-HA or Flag-CCDC134 bands; black arrow indicates putative C-terminal species of Gp96-HA. (E) Immunoblots of indicated knockout HEK293T cells. (F) Immunoblots of indicated CAL-1 cells. (G) HSP90B1 (gene coding for Gp96) mRNA levels of indicated CAL-1 cells measured by qPCR (normalized to HPRT1 ). (H) Immunoblots of indicated knockout CAL-1 cells treated with tunicamycin (Tun.) (5 µg/ml, for 0–4 h) or vehicle DMSO. In A, C–F, and H, data are representative of two independent experiments. In G, data show mean ± SD of four independent experiments. Source data are available for this figure: .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9888/pmc11629888/pmc11629888__jem_20240825_figs3.jpg)
![CCDC134 controls Gp96-dependent maturation and stability of plasma membrane and endolysosomal TLRs. (A) TNFα production of indicated Hoxb8-macrophages stimulated for 24 h with Pam3CSK4 (Pam3.) (0.1 μg/ml), LPS (10 ng/ml), R848 (0.1 μg/ml), CpG-B (ODN1668, 1 μM), or flagellin (0.1 μg/ml). Untr.: untreated. (B–D) Immunoblots of indicated Hoxb8-macrophages untreated (B) or stimulated with R848 (0.1 μg/ml), CpG-B (ODN1668) (1 μM) for 0–1 h (C) or Pam3CSK4 (0.1 μg/ml), LPS (10 ng/ml), and flagellin (0.1 μg/ml) for 0–1 h (D). (E and F) Immunoblots of indicated Hoxb8-macrophages. short exp.: short exposure; Mat.: mature; Imat.: immature, F: full length; C: cleaved form. (G) Quantification of TLR2, 4, 5, or 7 protein levels in sg Ren or sg CCDC134 Hoxb8-macrophages measured by surface (left upper panel) or surface and intracellular (intra.) (left lower panel) staining and quantified by gMFI (relative to respective sg Ren ). Representative histograms of surface (right upper panel) or intracellular (right lower panel). gMFI surface staining normalized to sg Ren TLR2 P value = 0.0053, TLR4 P value = 0.0063, TLR5 P value = 0.0093; gMFI surface and intracellular staining normalized to sg Ren TLR2 P value = 0.0002, TLR4 P value = 0.0256, TLR5 P value = ns, TLR7 P value <0.0001, two-tailed one sample t test. (H) Immunoblots of lysate from sg Ren or sg CCDC134 Hoxb8-macrophages treated with EndoH (H) or PNGase F (F). Mat.: mature form; Imat.: immature form; dg: deglycosylated. (I) Representative image of sg Ren or sg CCDC134 Hoxb8-macrophages stained for TLR7 (scale bar: 5 μm) (left panel) and quantification of TLR7 intensity (right panel). Data are expressed as fold change (FC) and pooled from n = 2. Violins show the variation of individual cells across all experiments; P value <0.0001, two-tailed Mann–Whitney test. (J) Quantification of CD11a, CD18, CD49d, and CD44 protein levels in sg Ren or sg CCDC134 Hoxb8-macrophages measured by surface (upper panel) or surface and intracellular (intra.) (bottom panel) staining and quantified by gMFI (relative to respective sg Ren ). Data show mean ± SD from three independent experiments; gMFI surface staining normalized to sg Ren CD11a P value = 0.0316, CD18 P value = 0.0449, CD49d P value = ns, CD44 P value = ns; gMFI surface and intracellular staining normalized to sg Ren CD11a P value = 0.0074, CD18 P value = ns, CD49d P value = 0.0029, CD44 P value = ns; two-tailed one sample t test. (K) Volcano plot of quantified proteins in whole proteome of sg CCDC134 versus sg Ren Hoxb8-macrophages (upregulated: red, fold change [FC] > 2 and P value <0.01; downregulated: blue, FC less than −2 and P value <0.01). pink: CCDC134 and Gp96; light pink: Bip and integrins; purple: TLRs; light purple: related to TLRs; green: IFN-stimulated gene proteins (ISGs). In B–F and H, data are representative of two experiments. In A, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. In G–J, data show mean ± SD from four or three independent experiments. Source data are available for this figure: .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9888/pmc11629888/pmc11629888__jem_20240825_fig6.jpg)
