Mper lysis buffer

After 22 h of incubation of HEK293T cells in 12‐well plates, HBSS and M‐PER™ lysis buffer (Thermo Fisher Scientific #78501) and Halt™ Protease‐Inhibitor‐Cocktail (Thermo Fisher Scientific #78441) were stored on ice. After 24 h of incubation, cells were rinsed with ice‐cold 1× HBSS (Sigma‐Aldrich #55037) and in each well of the 12‐well plate the HBSS was replaced with 200 μl M‐PER™ lysis buffer. The plate was kept on ice and gently shaken for 5–15 min. Then, the lysate from each well was transferred to a 1.5‐mL tube and centrifuged at 14,000× g for 15 min. Finally, 50 μl of each supernatant was transferred to a 96‐well plate (Sarstedt #83.3924.300). Fluorescence intensity was measured on a TECAN Spark 20 M plate reader. To measure GFP intensity, an excitation wavelength of 473 nm was used with 20 nm bandwidth, and an emission wavelength of 518 nm was used with 20 nm bandwidth. Per measurement, 30 flashes were performed with an integration time of 40 μsec. To measure mCherry intensity, an excitation wavelength of 565 nm was used with 20 nm bandwidth, and an emission wavelength of 610 nm was used with 20 nm bandwidth. Per measurement, 30 flashes were performed with an integration time of 40 μsec. All conditions for each biological replicate were measured at the same time on the same 96‐well plate. A fixed z‐position was used to measure each well of the 96‐well plate.