Tosyl lysyl chloromethyl ketone

Cell lines expressing reporters were cultured (1 × 10⁶ cells/ml) with dimethyl sulfoxide alone or with MG132 (Sigma-Aldrich; 25 μM) for 2 h. The cells were harvested, hypotonically lysed (10⁹ cells/ml) in distilled H₂O plus protease inhibitor cocktail and then adjusted to 1× TEN buffer conditions. Cytosolic and membrane fractions were separated by centrifugation (17,000 × g for 10 min at 4°C). Total, cytosolic, and membrane fractions (10⁷ cells equivalents) were processed for standard SDS–PAGE and immunoblotting. For protease protection, cytosolic and membrane fractions were prepared in the same manner, substituting TLCK (tosyl-lysyl-chloromethyl-ketone; Sigma-Aldrich). Samples (10⁷ cells equivalents, 25 μl) were treated (37°C, 30 min) with Proteinase K (Promega; 50 µg/ml final) in 1× TEN buffer with 10 mM CaCl₂ and in the presence or absence of 0.1% NP40 (final volume 40 μl). The enzyme was inhibited by addition of phenylmethylsulfonyl fluoride (PMSF, 5 mM final; Sigma-Aldrich) on ice for 5 min. Samples were then processed for standard SDS–PAGE and immunoblotting.