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. 2018 Apr 9;115(17):E4051–E4060. doi: 10.1073/pnas.1801340115

Fig. 3.

Fig. 3.

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.