FIT2 interacts with tubule-forming proteins and septins. (A) IP of FIT2-HA in FIT2-HA KI HepG2 cells transfected with control or siFIT2 RNA. Cells were lysed in 1% digitonin-containing buffer. IP was performed with anti-HA antibodies. The samples were analyzed by immunoblotting (IB) with the indicated antibodies. (B) Verification of FIT2-HA KI U2OS cells by Western blotting. (C) coIP of FIT2 and ER tubule-forming proteins in U2OS cells. IP was performed as in A. Samples were analyzed by immunoblotting with antibodies of different ER membrane proteins. (D) coIP of FIT2 and Flag-Rtn3a. WT and FIT2-HA KI HepG2 cells were transfected with Flag-Rtn3a. IP was performed as in A with anti-HA antibodies. The asterisk indicates nonspecific bands. (E) coIP of FIT2 and Flag-Rtn2. (F) coIP of FIT2 and Flag-Arl6IP. The asterisk indicates nonspecific bands. (G) coIP of FIT2-HA and ACSL3. HEK293T cells were transfected with FIT2-HA or empty vector and lysed in 1% Triton-containing IP buffer. IP was performed with anti-HA antibody in 0.5% Triton-containing IP buffer. Samples were analyzed by Western blotting with anti-HA and anti-ACSL3 antibodies. (H) coIP of FIT2-HA and HT008-Myc. As in G, except with cells coexpressing FIT-HA and HT008-Myc. (I) coIP of endogenous FIT2-HA and tubule-forming proteins in FIT2-HA KI U2OS cells transfected with siControl or siSeipin. (J) coIP of overexpressed FIT-HA and FIT2 H155A/H214A mutant (FIT2-DM-HA) with ER tubule-forming proteins in HEK293T cells. (K) Schematic diagram of the FIT2 constructs used. (L) Immunofluorescence assay detected the subcellular location of the indicated FIT2 constructs. Scale bar, 20 µm. (M) 293T cells were transfected with FIT2-HA or HA-vector and then treated with nocodazole or cytochalasin D. The coIP assay was performed as in Fig. 4 A. (N) As in M, but coIP of exogenous FIT2-HA or FIT2-DM-HA with endogenous septin 2 and 7. siControl, small interfering Control; siFIT2, small interfering FIT2; siSeipin, small interfering seipin.