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. 2010 Apr 26;285(29):22426–22436. doi: 10.1074/jbc.M110.123786

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 5. — MLK3 binds and stabilizes TRB3 protein. A, in vitro protein-protein interaction using bacterially expressed GST-MLK3-WT (lanes 1 and 3) and GST-MLK3-KD (lanes 2 and 4) to pull down in vitro transcribed and translated (IVT) HA-TRB3 (lanes 3–5) or reticulocyte lysate programmed with empty vector (lanes 1 and 2), followed by Western immunoblotting (IB) for TRB3 (top). Total input of MLK3 as Coomassie-stained proteins (middle) and their phosphorylation status (bottom) are shown. B, TRB3 and MLK3 interactions were assessed from lysates of cells co-transfected with HA-TRB3-WT (lanes 2, 3, and 5) and either GST-MLK3-WT (lanes 1–3) or GST-MLK3-KD (lanes 4 and 5) by GST pull-down (PD) of cellular extracts (PD), followed by Western blotting. Blots of GST pull-downs (panels i and iii) or 7.5% input (panels ii, iv, and v) using antibodies against pMLK3 (panel i), TRB3 (panels iii and iv), total MLK3 (panel ii), and co-transfected control FLAG-β-tubulin (panel v) are shown. Lane 3, effect of 60′ treatment with MLK inhibitor CEP11004. C, MLK3 interacts with the N terminus of TRB3. Shown are GST pull-downs from cells co-transfected with GST-MLK3-WT (lanes 1–4) and HA-TRB3-WT (lane 1) or TRB3 mutants ΔN94, ΔN201, ΔC310 (lanes 2–4, respectively), followed by Western blotting using anti-MLK3 (top) and anti-HA (middle) antibodies. Expression levels of TRB3 constructs were assessed using 7.5% input of total cellular lysate using anti-HA antibodies (bottom). D, schematic of TRB3 deletion mutants. E, time-dependent cycloheximide chase of HA-TRB3-WT (lanes 1–4) and HA-TRB3-Δ94 (lanes 5–8) immunoprecipitated using anti-HA beads from cells co-expressing GST-MLK3-WT followed by Western blotting using anti-MLK3 and anti-HA antibodies. Co-transfected FLAG-β-tubulin was used as a stable control. F, densitometric quantification of time-dependent TRB3 loss to determine t_½ of TRB3-WT (closed circles; t_½ = 3 h) or TRB3-Δ94 (open circles; T_½ = 45 min) in the presence of catalytically active MLK3.