PES1 Phosphorylation by CDK5/p25

Wild-type Flag-PES1 and mutant Flag-PES1 (S424A) proteins were translated in vitro following the manufacture protocol of TNT® Quick coupled Translation System Technical (Promega, Cat No. TM045) as described previously [22 (link)]. These proteins were purified with Pierce Protein G Agarose and primary antibody (Flag-tag antibody, Cat No. A5712, Bimake) in the cold room overnight. Then, the purified proteins were added into kinase assay buffer (Cat No.ab189135, Abcam), and incubated with activated CDK5/p25 (Cat No. ab60761, Abcam) and 50 μM ATP-γ-S (Cat No. ab138911, Abcam) at 30 °C for 45 min. 2.5 mM PNBM/5% DMSO were added to the sample at the room temperature for 1 h. The phosphorylated protein was detected by an anti-thiophosphate ester antibody (Cat No. ab92570, Abcam) [23 (link)].