Phosphorylation of SAP97/hDlg in HEK293 cells after cellular stress. Cells were incubated for 1 hour with or without 10 μM SB203580 and/or 5 μM PD184352, then exposed for 15 min to 0.5 M sorbitol or to UV C radiation (200J/m2), followed by a 30 min incubation. Endogenous SAP97/hDLg was immunoprecipitated from 1 – 5 mg of cellular lysate using 4 μg of anti-SAP97 (S552B), the resulting pellets were immunoblotted using antibodies that recognise SAP97 phosphorylated on serine 158 (anti-SAP97 phospho Ser 158 S285B), threonine 209 (anti-SAP97 phospho Thr 209 S938A), serine 431 (anti-SAP97 phospho Ser 431 S938A), serine 442 (anti-SAP97 phospho Ser 442 S965A) and an antibody that recognises unphosphorylated and phosphorylated SAP97 equally as well (anti-SAP97 S552B). The lanes in the panel are in duplicates. 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).