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Fig. 3 | Molecular Cancer

Fig. 3

From: SPOP-mediated ubiquitination and degradation of PDK1 suppresses AKT kinase activity and oncogenic functions

Fig. 3

SPOP ubiquitinates and subsequently degrades PDK1 in a degron dependent manner. A A schematic presentation of the evolutionarily conserved putative SPOP binding motif in PDK1. B IB analysis of WCL and IP products derived from HEK293T cells transfected with indicated constructs. Cells were treated with MG132 (10 μM) for 10 h before harvested. C IB analysis of WCL derived from HEK293T cells transfected with Flag-PDK1 (WT, Δ6S) and with/without HA-SPOP. D-E IB analysis WCL derived from HEK293T cells transfected with Flag-PDK1 (WT, Δ6S). Where indicated cells were treated with CHX (100 μg/ml) for the indicated time points before harvested. PDK1 protein abundance in (D) was normalized and quantified (E) (mean ± SD, n = 3) (t test), *P < 0.05, **P < 0.01. F IB analysis of WCL and His pulldown products derived from HEK293T cells transfected with indicated constructs. Cells were treated with MG132 (10 μM) for 12 h before harvested. G-I IB analysis of DLD1-PDK1 knockout cells stably infected with indicated constructs (G). Resulting cells were subjected to colony formation and soft agar assays (H), and the relative colony numbers were quantified (I) (mean ± SD, n = 3) (t test), **P < 0.01. J-L Cells generated in (G) were subjected to mouse xenograft assays. Tumor size was monitored (J), and dissected tumors were weighed (K, L). (mean ± SD, n = 8) (ANOVA test for J, and t test for L), *P < 0.05

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