Assessing NDRG1 Phosphorylation by GSK3β

Glycogen synthase kinase 3-beta (GSK3b) kinase inhibitor (SB216763) was purchased from Sigma-Aldrich. To determine the phosphorylation status of NDRG1, immunoprecipitation was used to pull down the NDRG1 protein, followed by an in vitro phosphorylation assay, similarly as previously described [57 (link),58 (link),59 (link)]. Briefly, Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, Dallas, TX, USA) with an anti-GFP antibody were used to precipitate NDRG1 proteins. After elution of the immunoprecipitated complex, the protein solution was subjected to an in vitro kinase assay by incubation with recombinant GSK3b protein (Sigma-Aldrich, St. Louis, MO, USA) in a solution containing ATP. Phosphorylated proteins were subjected to SDS-PAGE analysis, and the phosphorylation status of NDRG1 was determined by hybridization with an anti-phosphoserine antibody (Abcam, Cambridge, UK). Alternatively, NDRG1-FL- or NDRG1-Dc-transfected cells were treated with GSK3b-specific inhibitor (SB21673) at various doses for 24 h. Cellular proteins were extracted and subjected to a pulldown assay with an anti-GFP antibody. The phosphorylation status of NDRG1 was revealed by phospho-serine-specific immunoblotting.