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Briefly, following the indicated treatment, cells were washed twice with PBS and overlaid with 200 L of alkylation buffer (40 mM HEPES, 50 mM NaCl, 1 mM EGTA, protease inhibitors) supplemented with 25 mM N-ethylmaleimide (NEM

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The temperature should be controlled at 15-18 C, the air humidity was 90-95%, and the CO2 concentration was 0.03% (no special treatment was required for bud pressing)

Vps39 is the vacuole fusion factor responsible for vCLAMP contact sites [81]

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