(A) Logarithmically growing HTZ1(T46C)FL cells were incubated with or without 4-DPS (180 µM) before TCA fixation and protein extraction with the TUNES buffer with or without NEM (50 µM). Solubilized total proteins were dialyzed against two changes of 1 L TUNES buffer without NEM using the Slide-A-Lyzer button (10,000 Da MWCO, Thermo Fisher) at room temperature. Alexa647 maleimide (Thermo Fisher) was added to each reaction to a final concentration of 100 µM and was incubated at room temperature for 1 hr before analyzed by reducing SDS-PAGE and fluorescent densitometry. (B) Same as (A), except that the extracted proteins (before the dialysis step) were analyzed by non-reducing SDS-PAGE and anti-FLAG immunoblotting. (C) The V5HTA1(N39C) HTZ1(T46C)FL cells were treated with 4-DPS (180 µM) for 20 min before TCA fixation as described in Figure 1D. But after the addition of the TUNES buffer (with NEM), aliquots of were removed at the indicated times. The extracted proteins were analyzed by non-reducing SDS-PAGE and anti-FLAG western. (D) Band intensities of AxZ, ZxZ, and mono Z in (C) were plotted as a function of extraction time.