Fig. 5: Membrane-bound, but not soluble MIC, preferentially enhances NK cell cytotoxicity and associated signaling pathways.

a Cytotoxicity of human NKL cell line cultured with labeled target cells C1R, C1R-sMICA, C1R-mMICA at different E/T ratios for 4 h, as assessed by flow cytometry analyses of 7-ADD for tumor cell death. b Cytotoxicity of primary human NK cells against C1R-mMICA with stimulation conditions of sMICA (100 ng/ml) alone or in presence of B10G5 (5 µg/ml) assessed by lactate dehydrogenase (LDH) release. Immunoblot analysis to evaluate the activation of (c–e) Vav1 phosphorylated at Tyr 160 (p-Vav1) and PLCγ2 phosphorylated at Tyr 1217 (p- PLCγ2); f, g SLP-76 phosphorylated at Tyr 128 (p-SLP-76); and h–j ERK phosphorylated at Tyr 202/Tyr204 (p-ERK) and JNK phosphorylated at Thr183/Tyr185 (p-JNK) in human NK cells at the indicated condition and timepoints. Phosphorylated proteins were normalized to respective total proteins. Individual phospho-proteins, their respective total proteins, and β-actin were presented from the same blots. The blots were first probed for phospho-proteins, followed by stripping and re-probing for respective total proteins and subsequently for β-actin. The numbers represent quantified values after normalization and are represented as relative to NK only control conditions. The blots shown are representative of three independent experiments. The bar graphs show the quantitative data of relative protein levels and are represented as fold change over NK cell controls. All phosphorylated proteins were normalized to total proteins. Quantification was performed using Image J software. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (Student’s t test; two-tailed).