IP-MS identifies FAM49B interacting protein Rac.

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IP-MS identifies FAM49B interacting protein Rac. IP-MS identifies FAM49B interacting protein Rac. (A) Construction FAM49B with N-terminal triple FLAG mNeonGreen or C-terminal mNeonGreen triple Flag. (B) J.FAM49B cells were reconstituted with N-tagged or C-tagged FAM49B at an MOI of 0.5. The function of N-tagged or C-tagged FAM49B function were assessed by using CD69 inhibition index, which was calculated by MFI of CD69 in the GFP− population (nontransduced J.FAM49B cells) divided by that of GFP+ population (J.FAM49B cells reconstituted with FAM49B variants). ***P < 0.0001, *P < 0.05 (Student’s t test). (C) Coomassie blue staining of immunoprecipitated samples. An asterisk (*) indicates heavy chain and light chain of IgG antibody. (D) MS data showed ATPAF1, Rac1/2, and THEMIS peptides were enriched in the C-tag, but not N-tag or nontag FAM49B sample. (E) GST pull-down assay confirming the direction of FAM49B and Rac. Recombinant purified FAM49B protein was incubated with GST-Rac1 (in the presence or absence of GTPγS) or GST-Rac (G12V) protein for 1 h and IP with GST beads. The elutates from the GST beads and input samples were analyzed by Western blot. Data are representative of three independent experiments. IB, immunoblot. Wanjing Shang et al. PNAS 2018;115:17:E4051-E4060 ©2018 by National Academy of Sciences