HEK293 cells were exposed to an osmotic shock for the times indicated. SAKS1 was immunoprecipitated from the cell extract using 1 μg anti-SAKS1 (S ) 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 SAKS1 phosphorylated at Ser 200 (anti-SAKS1 phospho Ser 200 S747A) and with an antibody that recognises the phosphorylated and unphosphorylated forms of SAKS1 equally well (anti-SAKS1 S736A). 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).