Mechanism of TM₅₃₆ release from cells. (A) CM from TM₅₃₆-overexpressing cells were left untreated (−) or deglycosylated with EndoH or PNGaseF (+), then TM was detected by immunoblot. Enzyme’s ability to deglycosylate TM₅₃₆ was confirmed using TM₅₃₆ from the cell lysate. (B) The indicated forms of TM₅₃₆ were overexpressed in HEK293 cells, then TM was measured by ELISA in the CM and cell lysates (CM/lysate ratio reflects TM release capacity). The inset shows the hydropathy scores (ΔG) of IMD. Values are mean ± SD, expressed as fold change compared with TM₅₃₆. Statistical analyses were performed using analysis of variance (ANOVA) followed by Dunnett multiple comparison tests (vs TM₅₃₆): ∗P < .05; ∗∗∗P < .001. (C) Immunoblot detection of soluble forms of TM_536C527S contained in the CM of HEK293 cells overexpressing the inactive (−) or active form (+) of RHBDL2. (D) GPMV was prepared from HEK293 cells overexpressing TM_WT and TM₅₃₆ and incubated for 5 hours. TM in CM (sTM) and GPMV lysates (mTM) was measured by ELISA. The release of TM from GPMVs is shown by the ratios of sTM/mTM from 3 independent experiments (Exp 1-3). (E) Intracellular vesicles prepared from HEK293 and HELA cells overexpressing TM_WT and TM₅₃₆ were disrupted by incubation in Na₂CO₃ or saponin, and soluble TM (sTM) and mTM levels were measured by ELISA. The release of TM in the lumen of intracellular vesicles is shown by the ratios of sTM/mTM from 5 independent experiments (Exp 1-5). Exp, experiment; mTM, membrane TM.