Expression and release of TM₅₃₆. TM_WT and TM₅₃₆ were overexpressed in HEK293 cells then (A) TM was measured by ELISA in cell CM after 17 hours of accumulation and in cell lysates (CM/lysate ratio reflects TM release capacity) and (B) TM cell-surface expression was measured by flow cytometry. (C) Cells were homogenized for DRM preparation, and TM and Cav1 (DRM marker) were detected by immunoblot in each fraction of the sucrose density gradient. Densitometric analysis of the immunoblots (right panels) shows TM and Cav1 distribution in the gradient as a percentage of their total amount. (D) Immunoblot detection of TM and endogenous IR in lysates of cells overexpressing TM_WT and TM₅₃₆ and on their surfaces after biotinylation and enrichment of cell-surface–exposed proteins (d- indicates the position of TM homodimers). The enrichment of cell-surface–exposed proteins is attested by the detection of IR. The proIR is intracellular and only detected in cell lysates, whereas its mature form (IR) is exposed at the cell surface. (E) Lysates from cells overexpressing TM_WT or TM₅₃₆ were left untreated (−) or deglycosylated with EndoH (+), and TM was detected using immunoblot. Values are mean ± standard deviation (SD) expressed as fold change relative to TM_WT. Statistical analyses were performed using unpaired t test in panels A,B. ∗∗∗P < .001. CaV1, Caveolin 1; IR, insulin receptor; proIR, proform of IR.