Interactions between tubule-forming proteins and FIT2. (A) IP of FIT2-HA in WT and FIT2-HA KI HepG2 cells. WT and FIT2-HA KI 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) coIP of FIT2 and ER tubule-forming proteins in HepG2 cells. IP was performed as in A. Samples were analyzed by IB with antibodies of different ER membrane proteins. (C) FIT2-HA and REEP5-Myc were cotransfected into HEK293T cells and solubilized in triton-containing buffer or transfected individually into cells in different dishes, followed by mixing of the Triton-solubilized cell extracts. IP was performed with anti-HA or anti-Myc antibodies. (D) As in C, but with cells expressing FIT2-HA and/or GFP-Rtn4a. The asterisk (*) indicates degraded GFP-Rtn4a. (E) coIP of FIT2 truncations and Rtn4/REEP5. HEK293T cells expressing FIT2-HA, FIT2 truncations, or empty vector were lysed in 1% Triton-containing buffer and cell lysates immunoprecipitated with anti-HA antibodies.