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: 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: Reverse transcription reaction of 1 μg total DNA-free RNA was carried out using the iScript cDNA Synthesis Kit (1708891, Bio-Rad Laboratories) and RT-qPCR was performed on StepOne Plus System (Applied Biosystem, ThermoFisher Scientific) using the following Taqman gene expression assays: MICA (Hs00741286_m1), MICB (Hs00792952_m1), and ULBP2 (Hs00607609_m1).

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: Reverse transcription reaction of 1 μg total DNA-free RNA was carried out using the iScript cDNA Synthesis Kit (1708891, Bio-Rad Laboratories) and RT-qPCR was performed on StepOne Plus System (Applied Biosystem, ThermoFisher Scientific) using the following Taqman gene expression assays: MICA (Hs00741286_m1), MICB (Hs00792952_m1), and ULBP2 (Hs00607609_m1).

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: Reverse transcription reaction of 1 μg total DNA-free RNA was carried out using the iScript cDNA Synthesis Kit (1708891, Bio-Rad Laboratories) and RT-qPCR was performed on StepOne Plus System (Applied Biosystem, ThermoFisher Scientific) using the following Taqman gene expression assays: MICA (Hs00741286_m1), MICB (Hs00792952_m1), and ULBP2 (Hs00607609_m1).

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: Reverse transcription reaction of 1 μg total DNA-free RNA was carried out using the iScript cDNA Synthesis Kit (1708891, Bio-Rad Laboratories) and RT-qPCR was performed on StepOne Plus System (Applied Biosystem, ThermoFisher Scientific) using the following Taqman gene expression assays: MICA (Hs00741286_m1), MICB (Hs00792952_m1), and ULBP2 (Hs00607609_m1).

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: Reverse transcription reaction of 1 μg total DNA-free RNA was carried out using the iScript cDNA Synthesis Kit (1708891, Bio-Rad Laboratories) and RT-qPCR was performed on StepOne Plus System (Applied Biosystem, ThermoFisher Scientific) using the following Taqman gene expression assays: MICA (Hs00741286_m1), MICB (Hs00792952_m1), and ULBP2 (Hs00607609_m1).

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: Reverse transcription reaction of 1 μg total DNA-free RNA was carried out using the iScript cDNA Synthesis Kit (1708891, Bio-Rad Laboratories) and RT-qPCR was performed on StepOne Plus System (Applied Biosystem, ThermoFisher Scientific) using the following Taqman gene expression assays: MICA (Hs00741286_m1), MICB (Hs00792952_m1), and ULBP2 (Hs00607609_m1).

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: Reverse transcription reaction of 1 μg total DNA-free RNA was carried out using the iScript cDNA Synthesis Kit (1708891, Bio-Rad Laboratories) and RT-qPCR was performed on StepOne Plus System (Applied Biosystem, ThermoFisher Scientific) using the following Taqman gene expression assays: MICA (Hs00741286_m1), MICB (Hs00792952_m1), and ULBP2 (Hs00607609_m1).

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