anti ulbp2  (R&D Systems)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Breast cancer bone metastasis and bone metastatic cells retain NKG2DLs intracellularly: could this be a strategy to evade immune recognition?

doi: 10.3389/fcell.2026.1717607

Figure Lengend Snippet: MICA/B and ULBP2 show different localizations according to the tumor stage. (A) Representative immunohistochemical staining of MICA/B and ULBP2 is shown in adjacent normal tissues (ANT; n = 20), triple-negative breast cancer (TNBC; n = 33) and Luminal A type breast cancer (n = 47). Scale bar = 10 μm. (B,C) Mean score of the distribution of NKG2SLs in the cytosol/membrane fraction compared to the nuclei/perinuclear fraction in either TNBC or Luminal A breast cancer. (D) Representative immunohistochemical staining of MICA/B and ULBP2 is shown in bone metastasis (BoMet; n = 10) and paired bone metastatic ductal carcinoma (bmDC; n = 7). Scale bar = 10 μm. (E,F) Mean score of the distribution of NKG2SLs in the cytosol/membrane fraction compared to the nuclei/perinuclear fraction. Mean of scores derived from the semi-quantitative analysis for MICA/B and ULBP2. Each bar represents the mean ± SD of the specimens, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: For immunoblotting the following primary antibodies were used: MICA/B (F-6) (1:500, sc-137242, Santa Cruz Biotechnology), ULBP2 (1:1000, ab275023, AbCam), GM130 (1:10000, 12480, Cell Signaling Technology). β-Actin (1:2000, TA811000, Origene), was used as loading control for total cell lysates and cytoplasmic fraction, while TATA-binding protein (TBP, 1:1000, ab51841, AbCam), and Na + /K + -ATPase (1:20000, ab76020, AbCam) were used as loading controls for nuclear and membrane fractions, respectively.

Techniques: Immunohistochemical staining, Staining, Membrane, Derivative Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Breast cancer bone metastasis and bone metastatic cells retain NKG2DLs intracellularly: could this be a strategy to evade immune recognition?

doi: 10.3389/fcell.2026.1717607

Figure Lengend Snippet: MICA/B and ULBP2 are differentially expressed in BoMet and BC cell lines with different invasive potential. (A) mRNA expression levels of the NKG2DLs in different BC cell lines (1833, MDA-231, and MCF7) and non-tumorigenic breast cell line (MCF10A). Results are expressed as 2 −ΔΔCt compared to MCF7. ONE-way ANOVA: *p < 0.05; **p < 0.01; n = 3. (B) Western blotting analysis and bands intensity quantification of MICA/B and ULBP2 in BC cell lysates. The expression of MICA/B and ULBP2 was normalized on β-actin. Statistical analysis was performed using Kruskall-Wallis test for non-parametric distribution: *p < 0.05, n = 3.

Article Snippet: For immunoblotting the following primary antibodies were used: MICA/B (F-6) (1:500, sc-137242, Santa Cruz Biotechnology), ULBP2 (1:1000, ab275023, AbCam), GM130 (1:10000, 12480, Cell Signaling Technology). β-Actin (1:2000, TA811000, Origene), was used as loading control for total cell lysates and cytoplasmic fraction, while TATA-binding protein (TBP, 1:1000, ab51841, AbCam), and Na + /K + -ATPase (1:20000, ab76020, AbCam) were used as loading controls for nuclear and membrane fractions, respectively.

Techniques: Expressing, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Breast cancer bone metastasis and bone metastatic cells retain NKG2DLs intracellularly: could this be a strategy to evade immune recognition?

doi: 10.3389/fcell.2026.1717607

Figure Lengend Snippet: MICA/B and ULBP2 are expressed both on the surface and intracellularly in BoMet and BC cell lines with different invasive potential. (A) Surface staining of MICA/B and ULBP2 (green) in non-permeabilized breast cancer cells. Nuclei are stained with DAPI (blue). (B) Immunofluorescence detection of intracellular MICA/B and ULBP2 (green) in breast cancer cell lines. β-tubulin is marked in red; nuclei are stained with DAPI (blue). (C) Flow cytometry analysis of cell surface and intracellular MICA/B and ULBP2 expression. For surface staining cells were fixed with PFA and incubated with MICA/B-APC or ULBP2-PE primary antibody. For intracellular staining cells were fixed, permeabilized with 0.2% Tween-20, and incubated with primary antibody. Results are expressed as median fluorescence intensity (MFI) of each target after its isotype control subtraction. Intra-group statistical analysis was performed using ONE-way ANOVA with Tuckey’s multiple comparisons test: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n = 3.

Article Snippet: For immunoblotting the following primary antibodies were used: MICA/B (F-6) (1:500, sc-137242, Santa Cruz Biotechnology), ULBP2 (1:1000, ab275023, AbCam), GM130 (1:10000, 12480, Cell Signaling Technology). β-Actin (1:2000, TA811000, Origene), was used as loading control for total cell lysates and cytoplasmic fraction, while TATA-binding protein (TBP, 1:1000, ab51841, AbCam), and Na + /K + -ATPase (1:20000, ab76020, AbCam) were used as loading controls for nuclear and membrane fractions, respectively.

Techniques: Staining, Immunofluorescence, Flow Cytometry, Expressing, Incubation, Fluorescence, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Breast cancer bone metastasis and bone metastatic cells retain NKG2DLs intracellularly: could this be a strategy to evade immune recognition?

doi: 10.3389/fcell.2026.1717607

Figure Lengend Snippet: Intracellular MICA/B and ULBP2 co-localize with Golgi apparatus and show different mechanism of intracellular trafficking. (A) Subcellular localization of MICA/B and ULBP2 (green) in BC cell lines, and co-localization with calnexin (endoplasmic reticulum, ER) and GM130 (Golgi) (red). Nuclei are counterstained with DAPI (blue). White arrows indicate co-localization (yellow/orange staining). (B) Calnexin or GM130 co-localization with MICA/B or ULBP2 was quantified by ImageJ plug-in JACOP. Experiment was performed twice and analyzed in at least 6 fields for each condition. Statistical analysis was performed using Kruskall-Wallis for non-parametric distribution with Dunn’s multiple comparisons within organelles: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (C) Western blotting analysis of NKG2DLs and GM130 in subcellular fractions: cytosol (C), membrane (M), nucleus (N) and cytoskeleton (CS) extracts. TATA-binding protein (TBP), β-Actin and Na + /K + -ATPase were used as housekeeping for each fraction.

Article Snippet: For immunoblotting the following primary antibodies were used: MICA/B (F-6) (1:500, sc-137242, Santa Cruz Biotechnology), ULBP2 (1:1000, ab275023, AbCam), GM130 (1:10000, 12480, Cell Signaling Technology). β-Actin (1:2000, TA811000, Origene), was used as loading control for total cell lysates and cytoplasmic fraction, while TATA-binding protein (TBP, 1:1000, ab51841, AbCam), and Na + /K + -ATPase (1:20000, ab76020, AbCam) were used as loading controls for nuclear and membrane fractions, respectively.

Techniques: Staining, Western Blot, Membrane, Binding Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Breast cancer bone metastasis and bone metastatic cells retain NKG2DLs intracellularly: could this be a strategy to evade immune recognition?

doi: 10.3389/fcell.2026.1717607

Figure Lengend Snippet: MICA/B and ULBP2 are resistant to EndoH digestion but sensitive to PNGase F digestion. (A) Western blotting analysis of MICA/B and ULBP2 in cell lysates from BC cell lines, untreated or treated with 1500 U EndoH for 4 h. The bars represent the mean percentage of digested (□) and undigested (■) MICA/B and ULBP2 normalized on β-actin. Statistical analysis was performed using two-way ANOVA followed by Sidak’s multiple comparison test within cell line: *p < 0.05; **p < 0.001; ***p < 0.0001; n = 3. (B) Western blotting analysis of MICA/B and ULBP2 in cell lysates from BC cell lines, untreated or treated with 20 U PNGase F for 2 h. The bars represent the mean percentage of digested (□) and undigested (■) MICA/B and ULBP2 normalized on β-actin. Statistical analysis was performed using two-way ANOVA followed by Sidak’s multiple comparison test within cell line: **p < 0.001; ***p < 0.0001; n = 3.

Article Snippet: For immunoblotting the following primary antibodies were used: MICA/B (F-6) (1:500, sc-137242, Santa Cruz Biotechnology), ULBP2 (1:1000, ab275023, AbCam), GM130 (1:10000, 12480, Cell Signaling Technology). β-Actin (1:2000, TA811000, Origene), was used as loading control for total cell lysates and cytoplasmic fraction, while TATA-binding protein (TBP, 1:1000, ab51841, AbCam), and Na + /K + -ATPase (1:20000, ab76020, AbCam) were used as loading controls for nuclear and membrane fractions, respectively.

Techniques: Western Blot, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Breast cancer bone metastasis and bone metastatic cells retain NKG2DLs intracellularly: could this be a strategy to evade immune recognition?

doi: 10.3389/fcell.2026.1717607

Figure Lengend Snippet: NKG2DLs glycosylation and surface localization are differentially affected by tunicamycin in metastatic and non-metastatic BC cells and depend on intact Golgi for intracellular retention. (A) Western blotting analysis and bands intensity quantification of MICA/B and ULBP2 in BC cells after treatment with 1500 U tunicamycin for 24 h. The bars represent the mean percentage of digested (□) and undigested (■) MICA/B and ULBP2 normalized on β-actin. Statistical analysis was performed using two-way ANOVA followed by Sidak’s multiple comparison test within cell line: ***p < 0.0001; or using two-way ANOVA followed by Tukey’s multiple comparison test between cell lines: °p < 0.05; °°p < 0.01; °°°p < 0.001; °°°°p < 0.0001; n = 3. (B) Flow cytometry analysis of cell surface MICA/B and ULBP2 expression after treatment with 1500 U tunicamycin for 24 h. For surface staining cells were incubated with MICA/B-APC or ULBP2-APC primary antibody. Representative histograms overlay are shown for each cell line. Results are expressed as relative median fluorescence intensity (MFI) of each target to its untreated control (NT). Statistical analysis was performed using ONE-way ANOVA with Sidak’s multiple comparisons test within cell lines: *p < 0.05; ***p < 0.001; n = 3. (C) Subcellular localization of MICA/B and ULBP2 (green) in BC cell lines, and co-localization with GM130 (Golgi) (red) after treatment with 2 µM monensin for 3 h. Nuclei are counterstained with DAPI (blue).

Article Snippet: For immunoblotting the following primary antibodies were used: MICA/B (F-6) (1:500, sc-137242, Santa Cruz Biotechnology), ULBP2 (1:1000, ab275023, AbCam), GM130 (1:10000, 12480, Cell Signaling Technology). β-Actin (1:2000, TA811000, Origene), was used as loading control for total cell lysates and cytoplasmic fraction, while TATA-binding protein (TBP, 1:1000, ab51841, AbCam), and Na + /K + -ATPase (1:20000, ab76020, AbCam) were used as loading controls for nuclear and membrane fractions, respectively.

Techniques: Glycoproteomics, Western Blot, Comparison, Flow Cytometry, Expressing, Staining, Incubation, Fluorescence, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Breast cancer bone metastasis and bone metastatic cells retain NKG2DLs intracellularly: could this be a strategy to evade immune recognition?

doi: 10.3389/fcell.2026.1717607

Figure Lengend Snippet: Schematic representation of MICA/B and ULBP2 maturation and trafficking through Golgi apparatus in metastatic and non-metastatic BC cell lines. In metastatic cell lines (1833 and MDA-231), following protein synthesis, NKG2DLs acquire complex N-glycans and traffic from the endoplasmic reticulum (ER) to the Golgi apparatus. When N-glycosylation is impaired (e.g., by tunicamycin treatment), NKG2DLs fail to reach the cell surface. In contrast, in the non-metastatic MCF7 cell line, which is largely resistant to tunicamycin, immature NKG2DLs are still transported through the Golgi apparatus and delivered to the plasma membrane, resulting in increased surface expression irrespective of correct glycosylation. Nevertheless, regardless of glycosylation status, delivery to the plasma membrane requires an intact Golgi architecture, as demonstrated by the loss of co-localization with a Golgi marker following monensin treatment. Created in BioRender. Lombardi, G. (2025) https://BioRender.com/jnrm8ui .

Article Snippet: For immunoblotting the following primary antibodies were used: MICA/B (F-6) (1:500, sc-137242, Santa Cruz Biotechnology), ULBP2 (1:1000, ab275023, AbCam), GM130 (1:10000, 12480, Cell Signaling Technology). β-Actin (1:2000, TA811000, Origene), was used as loading control for total cell lysates and cytoplasmic fraction, while TATA-binding protein (TBP, 1:1000, ab51841, AbCam), and Na + /K + -ATPase (1:20000, ab76020, AbCam) were used as loading controls for nuclear and membrane fractions, respectively.

Techniques: Glycoproteomics, Clinical Proteomics, Membrane, Expressing, Marker

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: A The t-Distributed stochastic neighbour embedding (t-SNE) plot showing clustering information from GSE163558 and GSE206785 . B Venn diagram showing the overlap between mRNA microarray, GSE163558 and GSE206785 , upregulated genes associated with ECM, and Immunology Database and Analysis Portal (ImmPort) database. C Gene Ontology (GO) analysis showing enriched pathway terms for genes in GSE163558 and GSE206785 . D ULBP2 expression in fibroblasts revealed a correlation with collagen biosynthesis, collagen fibril organisation, ECM-receptor interaction, and TGF-β signalling. E ULBP2 mRNA expression of GC and corresponding adjacent normal tissues analyzed by mRNA microarrays (n = 16). F Immunoblotting of ULBP2 in the tumors and corresponding adjacent normal tissues from GC patients (n = 6). G Quantification of ULBP2 expression by immunohistochemistry (IHC) in human tissue microarrays (TMAs) from 62 gastric patients. H Kaplan–Meier survival analysis of overall survival (OS). I Representative images of gastric tissues with high ULBP2 expression compared to low-expression tissues, stained by IHC, Masson, and Sirius Red stains. J Scatter plots of ULBP2 expression versus collagen deposition in the human gastric TMAs. Data are expressed as mean ± SEM (***p < 0.001).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Microarray, Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: A ULBP2 knockout efficiency in SNU-216 cells and MKN-45 cells assessed by flow cytometry (FCM). B Growth curves of ctrl cells and ULBP2 −/− SNU-216 and MKN-45 cells (n = 3). C Colony formation assay and the statistical results of ctrl cells and ULBP2 −/− SNU-216 and MKN-45 cells (n = 3). D Transwell invasion assay of ctrl cells and ULBP2 −/− SNU-216 and MKN-45 cells (n = 3). E Representative images and growth kinetics of ctrl organoids and ULBP2 −/− organoids (n = 3). F Tumor growth curves of NSG mice carrying wild-type, ULBP2 −/− MKN-45 cell line-derived xenografts (CDX) (n = 5). G Representative images of tumors, H&E staining, and Ki-67 IHC staining (n = 3). Data are expressed as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Knock-Out, Flow Cytometry, Colony Assay, Transwell Invasion Assay, Derivative Assay, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: A Heat map showing differentially expressed genes (DEGs) in ULBP2 −/− MKN-45 cells compared to wild-type cells. B Transcriptome KEGG analysis revealed 20 pathways that ULBP2 knockout had a significant effect on MKN-45 cells. C ULBP2 knockout exhibited the most pronounced impact on ECM-receptor interaction and TGF-β signalling pathway of GC. D – F Transcriptome GSEA analysis revealed that the differentially expressed genes were significantly enriched in the TGF-β signalling pathway ( D ), ECM-receptor interaction ( E ), and collagen formation ( F ). G mRNA expression levels of TGF-β1 in ctrl cells and ULBP2 −/− MKN-45 and SNU-216 cells (n = 3). H Western blotting of TGF-β1, phosphorylated SMAD2 ( p -SMAD2), and total SMAD2/3 (T-SMAD2/3) in ctrl cells and ULBP2 −/− MKN-45 and SNU-216 cells. Data are represented as mean ± SEM (*p < 0.05, ***p < 0.001).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Knock-Out, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: A Diagram of the experimental procedure. B Growth curves of CAFs cultured with supernatants from ctrl cells and ULBP2 −/− SNU-216 and MKN-45 cells (n = 3). C The diagram of co-culture system between CAFs and GC cells. D EdU assay showing that the CAF proliferation was significantly reduced in the co-culture system of ULBP2 knockout MKN-45 cells compared with wild-type cells (n = 3). E Frequency of Ki-67 positive cells in the co-culture system, as determined by FCM (n = 3). F , G Relative levels of TGF-β1 ( F ) and collagen ( G ) content in the co-culture system (n = 6). H Diagram of the co-culture system between CAFs and GC organoids. I Representative bright field, H&E, and α-SMA staining images of the CAFs and organoid co-culture system. Data are represented as mean ± SEM (ns, nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Cell Culture, Co-Culture Assay, EdU Assay, Knock-Out, Staining

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: A Schematic diagram of ULBP2 CAR. B FCM showing the transduction efficiency of CAR-T cells. C IF analysis of the binding of ULBP2 CAR-T cells (green) to MKN-45-mCherry cells (red). D Killing efficiency of MKN-45 cells by ULBP2 CAR-T cells, determined by LDH-based cytotoxicity assay (n = 3). E Protein levels of IFN-γ and granzyme B in the culture supernatant (n = 3). F Killing efficiency of ULBP2 −/− MKN-45 cells by ULBP2 CAR-T cells (n = 3). G Representative images of GC organoids and killing efficiency of organoids by ULBP2 CAR-T cells (n = 3). H Representative bright field, H&E, and α-SMA staining images of ULBP2 CAR-T cells, CAFs, and organoid co-culture system, along with quantification of α-SMA positive area (n = 3). Data are represented as mean ± SEM (ns, nonsignificant; ***p < 0.001).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Transduction, Binding Assay, LDH Cytotoxicity Assay, Staining, Co-Culture Assay

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: A Diagram of the experimental procedure. B , C Tumor growth ( B ) and survival ( C ) curves of the CDX mice treated with ULBP2 CAR-T, anti-PD-1, or ULBP2 CAR-T cells plus anti-PD-1 (n = 5). D Bioluminescent imaging of the CDX mice at different days post tumor inoculation. E Protein levels of IFN-γ and granzyme B in the serum of CDX mice at different weeks post tumor inoculation detected by ELISA (n = 3). F Protein levels of PD-1, LAG-3, and TIM-3 in tumor-infiltrating lymphocytes at day 21 post tumor inoculation in the CDX mice that received different treatments, as shown by FCM (n = 3). G Representative images of tumors, H&E staining, Ki-67 IHC staining, and CD8 IF staining in the CDX mice that received different treatments. H Quantification of Ki-67 (n = 3) and CD8 + T (n = 6) cells in the CDX at day 21 post tumor inoculation. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Imaging, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: A Diagram of the experimental procedure. B – D Tumor growth B and survival curves ( C , D ) of mice bearing the PDX from a patient treated with ULBP2 CAR-T or ctrl cells alone, or ULBP2 CAR-T cells plus anti-PD-1 (n = 5). E Representative images of H&E, Ki-67 IHC, CD8 IHC, Masson, and α-SMA IF staining in the PDX mice that received different treatments. F – I Quantification of Ki-67 F (n = 3), CD8 + T cells G (n = 6), collagen area H (n = 6), and α-SMA area (n = 3) in the PDX at day 21 post tumor inoculation. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Staining

Journal: Cell Death & Disease

Article Title: ULBP2 CAR-T cells enhance gastric cancer immunotherapy by inhibiting CAF activation

doi: 10.1038/s41419-025-07905-5

Figure Lengend Snippet: ULBP2 overexpression promotes TGF-β signalling, driving CAF activation, leading to a dense stromal microenvironment. Moreover, targeting ULBP2 effectively reduces stromal deposition, stimulates CAR-T cell infiltration, and augments the antitumor efficacy of PD-1 blockade therapy. This graphical abstract was created with BioRender.com (License #NP28FJISA5, 2025).

Article Snippet: The following primary antibodies were used: anti-human ULBP2 (13133-1-AP; Proteintech) and anti-human CD8 (ab316778; Abcam).

Techniques: Over Expression, Activation Assay