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. Author manuscript; available in PMC: 2020 Oct 17.
Published in final edited form as: Oncogene. 2019 Jan 21;38(19):3569–3584. doi: 10.1038/s41388-019-0690-0

Fig. 1.

Fig. 1

MLK3 and PAK1 association. a The structure of MLK3 and PAK1 domains. The five proline-rich regions within PAK1 is marked by blue lines. b Total cell lysates were prepared from HEK-293, expressing indicated plasmids and mammalian GST-PAK1 was pulled-down and blotted for M2-tagged MLK3. c M2-tagged MLK3 was immunoprecipitated from HEK-293 cells expressing indicated plasmids and blotted for associated GST-PAK1 by anti-GST antibody. d Endogenous PAK1 was immunoprecipitated from Jurkat cell lysates and blotted with anti-MLK3 antibody. Anti-IRS1 and normal IgG were used as controls. e The direct association between MLK3 & PAK1 was determined by incubating purified proteins in solution and MLK3 was pulled down with anti-MLK3 antibody and blotted with PAK1 antibody. Anti-GAPDH antibody pull down was used as control. f GST-tagged (mammalian) MLK3 deletion mutants were created by PCR cloning and their association with Myc-tagged PAK1 was determined by pulling down GST-MLK3 mutants. Interacting domains are represented schematically. g GST-tagged PAK1 (mammalian) deletion mutants were created by PCR cloning and co-transfected in HEK-293 cells along with M2-tagged MLK3. The association between mutants and MLK3 was determined by M2-MLK3 pull down and blotted for GST associated PAK1. Since the molecular size of GST-PAK1 (268-545 aa), lane1 (denoted*), was similar to the size of IgG heavy chain, the GST-PAK1 associated with Flag-MLK3 was eluted off the beads after immunoprecipitation using Flag (M2 peptide). Interacting domains are represented schematically.