PhosphoProtein Purification Kit (6)

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Publication protocol

Quantitaion of phosphorylated SET:Jurkat cells expressing GFP-SET-WT were stimulated by PHA and lysed using a lysis buffer supplied with a PhosphoProtein Purification Kit (Qiagen) according to the manufacturer's instruction. In some experiments, cells were treated under the presence of PKD2 inhibitor Gö6976 (3 µM) before and during the PHA stimulation or the cells were treated with siRNA as described below before the stimulation. The fluorescence of GFP in the lysate was measured at excitation 475 nm and emission 505 nm using a Hitachi Fluorescence Spectrometer F-2500 (Tokyo, Japan). Each lysate containing the same amount of fluorescence was applied to the PhosphoProtein Purification Columns (Qiagen). Phospho-GFP-SET-WT recovered was subjected to SDS-PAGE and transferred onto a PVDF membrane. The phospho-GFP-SET-WT was detected and quantified by anti-GFP antibody and horseradish peroxidase-conjugated secondary antibody followed by enhanced chemiluminescent detection (ECL, Amersham Biosciences).

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