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. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: J Immunol. 2011 Jan 26;186(5):2959–2969. doi: 10.4049/jimmunol.1000112

FIGURE 5. Activation of MAP kinases upon 2DL4 cross-linking in an NK cell line was unaffected by Triad3A expression.

FIGURE 5

KHYG-1 cells (harvested 2 days after fresh IL-2 culture) transduced to express FLAG-2DL4 ± FLAG-Triad3A were stimulated with soluble anti-FLAG (M2) mAb (10 μg/ml) for the indicated minutes or PMA and ionomycin (p/i) for 10 min. A, Cells were then lysed and aliquots of precleared lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-ERK (pERK), anti-phospho-p38, and anti-GAPDH. The results are representative of at least three independent experiments. B, JNK was assayed from the same cell lysates with an in vitro kinase assay using GST-c-Jun (1–79 aa) as a JNK-binding matrix and substrate. Stimulated lysates were adsorbed with GST-c-Jun on glutathione-sepharose and suspended with JNK assay buffer containing cold ATP at 30°C for 20 min. Reactions were terminated with 3X Laemmli buffer and analyzed by immunoblotting with anti-phospho-c-Jun. Unmanipulated lysates from same samples were immunoblotted with anti-pJNK and anti-GAPDH.