Jurkat cells were incubated for 1 hour without (-) or with (+) 10 μM SB 203580 and/or 2 μM PD 184352, then left untreated (-) or exposed (+) for 15 mins to either 10 μg/ml of the protein synthesis inhibitor anisomycin (uppermost panel) or HOS [0.5M sorbitol] (middle and lower panel). CapZIP was immunoprecipitated from the cell extract using 1 μg anti-CapZIP (S970A) per 0.5 mg of extract. The immunoprecipitate was denatured in SDS, subjected to SDS/PAGE and transferred to nitrocellulose membranes. The membranes were immunoblotted with an antibody that recognises CapZIP phosphorylated at Ser 179 and with an antibody that recognises the phosphorylated and unphosphorylated forms of CapZIP equally well (anti-CapZIP S970A). Binding of the primary antibody was detected using rabbit peroxidase conjugated anti- sheep IgG antibody (1 in 10, 000 dilution, Pierce) followed by enhanced chemiluminescence (ECL, Amersham).