Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 controls Gp96 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: .

Article Snippet: Plasmids coding for UNC93B1 (ID:132298), Gp96 (ID: 82130), TLR4 (ID: 13086), and TLR5 (ID: 13019) were from Addgene.

Techniques: Glycoproteomics, Transfection, Mass Spectrometry, Stable Transfection, Control, Transduction, Plasmid Preparation, Staining, Recombinant, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Knock-Out

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 interacts and stabilizes the ER chaperone Gp96. (A and B) Immunoprecipitates (IP) and whole-cell extracts (WCE) from transfected HEK293T (A) or knockout CAL-1 cell lines (B) as indicated. (C) Schematic of wildtype Gp96 and deletion constructs (left panel) as well as immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T transfected as indicated (right panel). SP: signal peptide, pN: pre-N-terminal domain; ND: N-terminal domain, CR: charged linker region; MD: middle domain; CD: C-terminal domain; KDEL: ER retention motif; steady state (N217 red) and cryptic N-glycans acceptor sites are shown. (D) Immunoprecipitates (IP) and input using Flag-CCDC134 and Gp96-HA recombinant proteins. For the complex formation, Flag-CCDC134 and Gp96-HA were preincubated overnight at 4°C before to perform the immunoprecipitation assay. Red arrow indicates specific Gp96-HA or Flag-CCDC134 bands; black arrow indicates putative C-terminal species of Gp96-HA; asterisks indicate IgG heavy or light chains. (E) Immunoblots of lysates from indicated knockout CAL-1 cells. Asterisks indicate a non-specific band. (F) IL-6 production of indicated knockout CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (G) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) from indicated CAL-1 cell lines. (H) Immunoblots from indicated knockout CAL-1 cells treated with NMS-873 (1, 5, and 10 μM) or vehicle DMSO for 8 h. In A–E, G, and H, data are representative of two independent experiments. In F, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: .

Article Snippet: Plasmids coding for UNC93B1 (ID:132298), Gp96 (ID: 82130), TLR4 (ID: 13086), and TLR5 (ID: 13019) were from Addgene.

Techniques: Transfection, Knock-Out, Construct, Recombinant, Immunoprecipitation, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: CCDC134 modulates TLR7/9 signaling by regulating Gp96 hyperglycosylation. (A) Immunoblots of knockout CAL-1 cells stably expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1 or 2) treated with the indicated concentration of doxycycline (Dox., 0–10 ng/ml) for 24 h. (B) Immunoblots of knockout CAL-1 cells stably expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1) treated with doxycycline (Dox., 5 ng/ml) for 24 h. (C) IL-6 production of indicated knockout CAL-1 cells expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1) induced with 5 ng/ml of doxycycline (Dox.) for 17 h and followed by 24 h stimulation with R848 (5 μg/ml). (D) Immunoprecipitates (IP) and whole-cell extracts (WCE) from TLR7-V5-expressing CAL-1 knockout cell lines as indicated. Asterisks indicate a non-specific band. (E) Immunoblots of lysates from indicated CAL-1 cell lines. long exp.: long exposure. (F) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (G) Immunoblots of indicated CAL-1 cells treated with R848 (5 µg/ml, for 0–1 h). (H) Immunoblots of cell lysates treated with EndoH (H) or PNGase F (F). N3Q Gp96 bears mutations at positions N445Q-N481Q-N502Q. (I) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). In A, B, D, E, G, and H, data are representative of two independent experiments. In C, F, and I, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: .

Article Snippet: Plasmids coding for UNC93B1 (ID:132298), Gp96 (ID: 82130), TLR4 (ID: 13086), and TLR5 (ID: 13019) were from Addgene.

Techniques: Western Blot, Knock-Out, Stable Transfection, Expressing, Construct, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: Mapping of CCDC134 requirement for regulation of Gp96 in the ER. (A) Doxycycline-inducible CCDC134 mRNA ( dox.-ind . CCDC134 ) levels in indicated cells measured by qPCR. The primers were specifically designed to detect only the doxycycline-inducible construct, excluding any detection of the endogenous CCDC134 mrRNA. CAL-1 cells stably expressing the doxycycline-inducible CCDC134 construct (clone 1 or 2) were treated with the indicated concentration of doxycycline (Dox., 0–10 ng/ml) for 24 h. (B) AlphaFold structure prediction for human CCDC134 (Uniprot entry name: Q9H6E4 CC134_HUMAN). (C) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) of indicated CAL-1 cells reconstituted with wildtype or deletion CCDC134 mutants. The Δ133–156 CCDC134 deletion mutant lacks the predicted N-linked glycosylated NQT sequon. Ev: empty vector. (D) Representative confocal microscopy images of HeLa cells transfected with Flag-tagged wildtype (WT) or deletion mutant CCDC134 constructs. Green: anti-Flag; red: anti-Calreticulin; blue: DAPI. Scale bar: 5 μm. (E) Representative confocal microscopy images of HeLa cells transfected with Flag-tagged Δ133–156 CCDC134 deletion mutant which lacks the predicted N-linked glycosylated NQT sequon (left panel) and quantification of the colocalization between Flag-CCDC134 (wildtype or Δ133–156 deletion mutant) and calreticulin (right panel). Data are expressed as colocalization score (coloc. score) and pooled from three independent experiments. Each dot represents analysis of a single field of view containing one to four transfected cells, and violins show the variation of individual dot across all experiments; P value = ns, two-tailed Mann–Whitney test. Green: anti-Flag; red: anti-Calreticulin; blue: DAPI. Scale bar: 5 μm. (F) Immunoblots of proteins from the (not precipitated) supernatant (SN) of indicated CAL-1 cells. Doxycycline-inducible CCDC134 cells (clone 1) were induced with doxycycline (5 ng/ml, Dox.) for 24 h. SH: strep-HA tag, ind. 134: doxycycline-inducible CCDC134. (G) Immunoblots of indicated CAL-1 cells incubated for 48 h with supernatants (SN) of donor cells cultured for 24 h in Opti-MEM. Doxycycline-inducible CCDC134 cells (clone 1) were induced with doxycycline (5 ng/ml, Dox.) for 48 h. SH: strep-HA tag, ind. 134: doxycycline-inducible CCDC134. In A, data show mean ± SD of three independent experiments. In C, F, and G, data are representative of two independent experiments. Source data are available for this figure: .

Article Snippet: Plasmids coding for UNC93B1 (ID:132298), Gp96 (ID: 82130), TLR4 (ID: 13086), and TLR5 (ID: 13019) were from Addgene.

Techniques: Construct, Stable Transfection, Expressing, Concentration Assay, Western Blot, Mutagenesis, Plasmid Preparation, Confocal Microscopy, Transfection, Two Tailed Test, MANN-WHITNEY, Incubation, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: CCDC134 controls TLR biogenesis through the ER chaperone Gp96

doi: 10.1084/jem.20240825

Figure Lengend Snippet: 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: .

Article Snippet: Plasmids coding for UNC93B1 (ID:132298), Gp96 (ID: 82130), TLR4 (ID: 13086), and TLR5 (ID: 13019) were from Addgene.

Techniques: Clinical Proteomics, Membrane, Western Blot, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Cell Stress & Chaperones

Article Title: Secreted extracellular heat shock protein gp96 and inflammatory cytokines are markers of severe malaria outcome

doi: 10.1016/j.cstres.2024.12.004

Figure Lengend Snippet: Circulating levels of Gp96 in plasma from malaria cases and controls. Comparison of plasma concentration of extracellular gp96 between (a) malaria cases and noninfected healthy subjects (controls) and (b) severe malaria, uncomplicated malaria, and control cases. Data expressed are transformed in logarithmic as mean ± SD. **When the difference between malaria cases and noninfected controls (healthy) was significant with P < 0.05. *When the difference between SM and UM was significant with P > 0.05.

Article Snippet: To quantify the gp96 protein in plasma, an ELISA kit supplied by Cloud-Clone Corp was used (Hsp90b1 ELISA kit (Euromedex)) according to the manufacturer’s instructions .

Techniques: Comparison, Concentration Assay, Control, Transformation Assay

Journal: Cell Stress & Chaperones

Article Title: Secreted extracellular heat shock protein gp96 and inflammatory cytokines are markers of severe malaria outcome

doi: 10.1016/j.cstres.2024.12.004

Figure Lengend Snippet: The correlation of gp96 with parasitémia and cytokines in SM, UM, and healthy control subjects.

Article Snippet: To quantify the gp96 protein in plasma, an ELISA kit supplied by Cloud-Clone Corp was used (Hsp90b1 ELISA kit (Euromedex)) according to the manufacturer’s instructions .

Techniques: Control

Journal: Nature Communications

Article Title: Heat shock protein gp96 drives natural killer cell maturation and anti-tumor immunity by counteracting Trim28 to stabilize Eomes

doi: 10.1038/s41467-024-45426-5

Figure Lengend 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).

Article Snippet: The mean fluorescence intensity of gp96 was analyzed by Imaris 9.7.

Techniques: Western Blot, Expressing, Flow Cytometry, Marker, RNA Sequencing, Produced, Two Tailed Test

Journal: Nature Communications

Article Title: Heat shock protein gp96 drives natural killer cell maturation and anti-tumor immunity by counteracting Trim28 to stabilize Eomes

doi: 10.1038/s41467-024-45426-5

Figure Lengend 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.

Article Snippet: The mean fluorescence intensity of gp96 was analyzed by Imaris 9.7.

Techniques: Marker, Expressing

Journal: Nature Communications

Article Title: Heat shock protein gp96 drives natural killer cell maturation and anti-tumor immunity by counteracting Trim28 to stabilize Eomes

doi: 10.1038/s41467-024-45426-5

Figure Lengend 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).

Article Snippet: The mean fluorescence intensity of gp96 was analyzed by Imaris 9.7.

Techniques: Expressing, RNA Sequencing, Flow Cytometry, Western Blot, Infection, Control, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: Heat shock protein gp96 drives natural killer cell maturation and anti-tumor immunity by counteracting Trim28 to stabilize Eomes

doi: 10.1038/s41467-024-45426-5

Figure Lengend 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).

Article Snippet: The mean fluorescence intensity of gp96 was analyzed by Imaris 9.7.

Techniques: Flow Cytometry, Immunostaining, Fluorescence, Staining, Confocal Microscopy, Western Blot, Transfection, Plasmid Preparation, Stable Transfection, Expressing, Knock-Out, Control, Double Knockout, Two Tailed Test

Journal: Nature Communications

Article Title: Heat shock protein gp96 drives natural killer cell maturation and anti-tumor immunity by counteracting Trim28 to stabilize Eomes

doi: 10.1038/s41467-024-45426-5

Figure Lengend 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.

Article Snippet: The mean fluorescence intensity of gp96 was analyzed by Imaris 9.7.

Techniques: Stable Transfection, Expressing, Transfection, Western Blot, Control, Plasmid Preparation, Flow Cytometry, Infection, Immunoprecipitation, Knock-Out, Ubiquitin Proteomics, Inhibition