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Fig. 2 | BMC Cancer

Fig. 2

From: Inhibition of neddylation facilitates cell migration through enhanced phosphorylation of caveolin-1 in PC3 and U373MG cells

Fig. 2

Neddylation antagonized the phosphorylation of caveolin-1 at Y14. a Ni-NTA-binding assay was performed in HEK293T cells co-expressing GFP-caveolin-1 (Cav1) and His-NEDD8 (N8) or His-NEDD8ΔGG (N8ΔGG). Neddylated proteins were pulled down with Ni-NTA beads under a denaturing condition, and subjected to Western blotting with the indicated antibodies. b HEK293T cells expressing FLAG tag or FLAG-NEDD8 were lysed and subjected to co-immunoprecipitation analysis. Precipitated and input proteins were analyzed by Western blotting. c Ni-NTA-binding assay was performed in HEK293T cells co-expressing His-NEDD8 and GFP-caveolin-1 or GFP-caveolin-1-K5R. Neddylated proteins were pulled down with Ni-NTA beads under a denaturing condition, and subjected to Western blotting with the indicated antibodies. d PC3, U373MG, and A549 cells were treated with MLN4924 (0.125, 0.25, or 0.5 μM) or DMSO (Veh) for 24 h and then subjected to Western blotting (top). pY14-Cav1 represents caveolin-1 phosphorylated at Y14 residue. The level of pY14-Cav1 along with the range of MLN4924 was quantified based upon the relative level of β-tubulin from PC3 and U373MG cell lysates (bottom). e A549 cells were treated with 0.5 μM MLN4924 and subjected to the wound-healing analysis for 24 h. Each bar represents the means + standard deviation of migration areas from three independent experiments, and n, s, denotes P > 0.05 between the indicated groups. Scale bar = 200 μm

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