Interactions between septins and FIT2. (A) HEK293T cells transfected with FIT2-HA or empty HA vector were lysed, and coIP assay was performed by incubating cell lysates with anti-HA agarose beads. Immunoblotting (IB) was performed using the indicated antibodies. (B) As in A, but with or without 5 mM GTP in the lysates. (C) FIT2 KI U2OS cells were cultured under normal conditions or LD biogenesis conditions (fatty acid starvation [Starv.] for 60 h, OA treatment for 15 min). After DSP-mediated protein cross-linking, coIP of FIT2-HA with endogenous septin 7 was performed using anti-HA agarose beads. (D) As in C, coIP of FIT2-HA and endogenous septin 2, 6, and 7 in the LD biogenesis condition. (E and F) Coflotation assay using FIT2-reconstituted proteoliposomes and purified septin2/6/7 hexamer (E) or individual septins (septin 2, 6, 7, and 9; F). Proteins in each fraction were detected by IB using the indicated antibodies. (G) In vitro peptide pull-down assays were performed using the indicated biotinylated peptides and purified septin2/6/7 hexamer, followed by SDS-PAGE and Coomassie blue staining. (H) HEK293T cells were transfected with the indicated FIT2-HA truncations, coIP and IB assays performed as in A. (I) BLI assays using biotinylated FIT2 NTL peptide-coated sensor and individually purified septin 2, septin 7, septin 7-ΔCC, and septin 7-CC at the gradient concentration of 31.2–500 nM. The dissociation constant (K_d) is shown. CTL, C-terminal loop; ctyL1, cytosolic loop 1; ctyL2, cytosolic loop 2.