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