O pdm

The following reagents were used. Cys-specific chemical crosslinkers: BMH and TMEA (Thermo Scientific), o-PDM and p-PDM (Sigma) and Bis-MTS (methanethiosulfonate) reagents (M1M through M17M) (Toronto Research Chemicals). Detergents: Brij L23, Brij 97 and Brij 99 (Sigma), Triton-X-100, NP-40 and Tween-20 (MP Biochemical) and Digitonin (Sigma and Calbiochem). o-phenanthroline (Wako). Polyene antibiotics: nystatin (Wako) and natamycin (Fluka). Other chemicals were purchased from Sigma, Wako Pure Chemical, Nacalai Tesque and BD.

HEK293T were plated in 6 wells plates and co-transfected with indicated constructs. TpoR L508C was truncated after Box 2 to avoid non-specific crosslinking of intracellular cysteines. 48 h post transfection, cells were harvested without trypsinization and washed in PBS. Cells were then re-suspended and incubated for 15 min at room temperature in crosslinking buffer (PBS 1 mM MgCl₂, 0.1 mM CaCl₂) with 100 μM N-ethylmaleimide (NEM, ThermoFisher Cat.:23030) to avoid non-specific crosslinking of extracellular cysteines. 200 ng/mL of rhTpo was added in the indicated condition. Samples were mixed gently and further incubated for 15 min at room temperature. Cells were then centrifuged for 5 min at 500×g and re-suspended in crosslinking buffer with 100 μM o-phenylene dimaleimide (o-PDM, Sigmaaldrich) for 10 min at room temperature. Cells were further centrifuged 5 min at 500×g and re-suspended in lysis buffer (NP-40, 2% ß-mercaptoethanol) with protease inhibitor cocktail. Cell lysates were analyzed by SDS-PAGE in denaturing and reducing conditions with anti-HA antibody.