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. Author manuscript; available in PMC: 2011 Aug 17.
Published in final edited form as: Oncogene. 2010 Oct 4;30(7):781–789. doi: 10.1038/onc.2010.453

Figure 4.

Figure 4

Merlin regulates MLK3 activity by inhibiting the Cdc42-MLK3 interaction. (a) HEK293 cells were transfected with FLAG-Cdc42 and nonspecific or merlin siRNA. GTP-bound FLAG-Cdc42 was isolated from cell lysates with Pak-1 binding domain (PBD) beads and detected with FLAG antibody. For control samples, cell lysates were incubated with GTP γS or GDP prior to the addition of PBD beads. Protein expressions were verified by Western blotting with the indicated antibodies. (b) HEK293 cells were transfected with FLAG-Cdc42 (wild-type, V12 or N17) with or without Myc-merlin. GTP-bound FLAG-Cdc42 was isolated from cells lysates with PBD beads and detected with FLAG antibody. Western blotting of cell extracts was performed with FLAG and Myc antibodies to verify protein expression. (c) FLAG-Cdc42 was overexpressed in RT4-NF2.17 cells. Cells were untreated or treated with 1.0μg/ml doxcycline for 24 h to induce merlin expression. FLAG-Cdc42 immunoprecipitates were analyzed by Western blotting with the indicated antibodies. (d) Myc-merlin, FLAG-Cdc42, and GST-MLK3 were overexpressed in HEK293 cells. FLAG-Cdc42 immunoprecipitates and cell lysates were analyzed by Western blotting with the indicated antibodies. (e) Myc-merlin, FLAG-Cdc42 (wild-type, V12, or N17), and HA-MLK3 were overexpressed in HEK293 cells. FLAG-Cdc42 immunoprecipitates and cell extracts were analyzed by Western blotting with the indicated antibodies. IB, immunoblot; IP, immunoprecipitate.