Figure 6. NDRG1 is an FBW7 substrate.
(A) MCF7 cells infected with SGK3 shRNA, Akt1 shRNA or control pLKO, serum-starved then stimulated with IGF-1 for 20 min. Cell lysates were immunoblotted with the indicated antibodies.
(B) MCF10A cells were treated with cycloheximide or control DMSO for the indicated times and cell lysates immunoblotted with anti-NDRG1 and anti-p85.
(C) T47D, MCF10A, MCF7, and ZR-75-1 cells were treated with MG132 for 16 hr and immunoblotted with anti-NDRG1 and anti-p85.
(D) Cell lysates of wild-type (Fbw7 WT) or DLD-1 colon cancer cells null for Fbw7 (Fbw7 −/−) were immunoblotted with the indicated antibodies.
(E) MCF7 cells were infected with two distinct Fbw7 shRNA’s or control pLKO, and lysates immunoblotted with the indicated antibodies.
(F) HEK293T cells were transfected with control GFP vector, alone or with the indicated combinations of NDRG1-FLAG, HA-Fbw7, HA-GSK3β.S9A and SGK3-GST. NDRG1 stability was evaluated by immunoblotting whole cell lysates with anti-FLAG, and indicated antibodies.
(G) HEK293T cells were transfected for 48 hr with NDRG1 -FLAG prior to 5 hr treatment with GSK650394, SB415286, GSK650394 and SB415286, or MG132 in order to examine NDRG1 protein stability.
(H) A His-Ubiquitin assay was performed after transfection of HEK293T cells with FLAG-NDRG1, GST-Fbw7, HA-GSK3β.S9A, SGK3-GFP, and His-Ub, followed by treatment with MG132 for 12 hr. Nickel NTA beads were used to isolate His-ubiquitin complexes, followed by immunoblotting with the indicated antibodies.
(I) ZR-75-1 cells transfected for 24 hr with pcDNA3, NDRG1, 8A–NDRG1, Fbw7, GSK-3β, and SGK3 followed by an 18 hr Transwell migration assay. Quantification of migrated cells and representative immunoblots of whole cell lysates (WCL) with indicated antibodies are shown. Error bars represent standard deviation. See also Figure S6.