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. 2010 Jan 8;285(11):8122–8129. doi: 10.1074/jbc.M110.101824

FIGURE 2.

FIGURE 2.

NLK phosphorylates FOXO1 in vivo and in vitro. A, COS1 cells were transfected with denoted plasmids. After 36 h of transfection, the cells were lysed for anti-HA immunoprecipitation (IP) assays. The immune complexes containing FOXO1 were incubated with λ-phosphatase or buffer alone and then subjected to immunoblot (IB) analyses using anti-HA antibody (top panel). Anti-HA and anti-FLAG blots were also completed for the same whole cell lysates (middle and bottom panels). B, COS1 cells were transfected with FLAG-NLK, GST-FOXO1 N1, N2, C1, and C2 constructs. After 36 h of transfection, the cells were lysed for GST pulldown assays. The precipitated proteins were subjected to immunoblot analyses using anti-GST antibody (top panel). Anti-FLAG blots were also completed for the same whole cell lysates (bottom panel). C, HEK293T cells were transfected with FLAG-NLK WT or KN constructs. Expressed proteins were immunopurified with anti-FLAG antibody, and in vitro kinase assays were performed using GST-FOXO1 C1 as a substrate. The phosphorylated FOXO1 C1 proteins were visualized by autoradiography (top panel), and the C1 protein levels were compared by Coomassie Blue staining (middle panel). FLAG-NLK WT and KN protein levels were visualized by anti-FLAG immunoblot (bottom panel). D, FLAG NLK WT, KN, and full-length FOXO1-HA constructs were separately transfected in HEK293T cells. After 36 h of transfection, the cells were lysed for anti-HA or anti-FLAG immunoprecipitation. The immune complexes for NLK and FOXO1 were mixed together as indicated to conduct in vitro kinase assays. The phosphorylated FOXO1-HA proteins were visualized by autoradiography (top panel). FOXO1-HA and FLAG-NLK protein levels were visualized by anti-HA (middle panel) and anti-FLAG (bottom panel) immunoblots, respectively. The asterisk indicates NLK-associated phosphorylation activity, which does not seem to be related to FOXO1 phosphorylation. E and F, control or NLK siRNA was transfected with (E) or without (F) FOXO1-HA construct in COS1 cells. After 72 h of transfection, the cells were lysed for immunoblot analyses using anti-HA, anti-FOXO1, anti-NLK, and anti-β-tubulin antibodies.