Bas 2500 phosphoimager

Immune complex kinase assays were performed as previously described^{52 (link),53 (link)}. Briefly, cells were lysed in buffer A (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 12 mM β-glycerophosphate, 5 mM EGTA, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 10 mM NaF, 1 mM dithiothreitol), and the soluble fractions (equal amounts of protein) of the lysates obtained by microcentrifugation were subjected to immunoprecipitation with appropriate antibodies. The resulting precipitates were incubated for 30 min at 30 °C in 15 μl of a kinase reaction buffer^{53 (link)} in the presence of 1 μCi of [γ-³²P]ATP and 2 μg of indicated substrate protein. Bacterially expressed GST fusion proteins of MKK6(K82A) and c-Jun(1–79) were used as substrates for ASK1 and JNK, respectively. The reaction mixtures were subjected to SDS-PAGE, and the extent of phosphorylation of the substrate proteins was analyzed with a Fuji BAS 2500 phosphoimager.

GST-CIB1 expressed in Escherichia coli was purified with the use of glutathione-conjugated agarose beads (Sigma). ASK1 variants were translated in vitro in the presence of [³⁵S]methionine with the use of a TNT reticulocyte lysate system (Promega). The ³⁵S-labled proteins were incubated at 4 °C for 4 h in a binding buffer^{54 (link)} with GST-fused proteins immobilized on glutathione-agarose beads. The bound ³⁵S-labeled proteins were eluted from the beads and analyzed by SDS-PAGE and with a Fuji BAS 2500 phosphoimager.