Characterization of released sTM₅₃₆. (A) HEK293 cells overexpressing TM_WT and TM₅₃₆ were treated with TMI-5 (+; 5 μM; 17 hours), and TM was measured by ELISA in CM and cell lysates. (B) TM_WT and TM₅₃₆ were overexpressed together with inactive (−) or active (+) RHBDL2, and TM was measured by ELISA, as in panel A (inset shows RHBDL2 expression in cell lysates; Ø, no RHBDL2). (C) CS-TM₅₃₆ was overexpressed together with inactive (−) or active (+) RHBDL2, and sTM₅₃₆ in CM was detected by immunoblot. (D) Immunoblot detection of TM₅₃₆ and Flot1 (used as a cell lysate marker) contained in CM and cell lysate. Values are mean ± SD, expressed as fold change relative to TM_WT-overexpressing cells. Statistical analyses were performed using an unpaired t test for panel A or Mann-Whitney test for panel B. ∗∗P < .01; ∗∗∗P < .001. CS-TM₅₃₆, CS-devoid TM₅₃₆; Flot1, Flotilin 1.