Roti blue quick protein stain

Constitutive 20S proteasome preparations (0.5 µg/track) (Enzo, Farmingdale, NY, USA) were loaded onto the 4-20% gradient polyacrylamide gel. The electrophoresis was performed for 36 h at +4 °C (at 80 V 12 h, 140 V 12 h, and 240 V 12 h). Following the electrophoresis, the gel was cut into slices. Then gel slices containing proteasome preparations were soaked with 6 mM of ATP or 3, 6 or 20 mM of Mg²⁺, or their combinations, together with 100 µM of Suc-LLVY-AMC substrate. After that, gel slices were incubated for 30 min and analyzed under UV. ImageJ software (https://imagej.net/software/fiji/ accessed on 12 June 2020) was used to analyze the acquired data. Gels were stained with ROTI^®Blue quick protein stain (Carl Roth, Karlsruhe, Germany) to confirm an equal amount of proteasomes.

The Me4BodipyFL-Ahx3Leu3VS (UbiQbio, Amsterdam, The Netherlands) proteasome activity probe was used to detect proteolytically active proteasome subunits using SDS-PAGE according to the described protocol [42 (link)]. Shortly, cellular lysates (app. 20 µg) were mixed with 0.5 µL of probe in PBS and were incubated for 1 h at 37 °C. Then samples were loaded into 15% Tris-Glycine polyacrylamide gel and following the electrophoresis BodipyFL fluorescence was analyzed at the excitation wavelength 480 nm and emission wavelength 530 nm using ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA). After that, the gel was stained with ROTI^®Blue quick protein stain (Carl Roth, Karlsruhe, Germany) to ensure an equal protein load.

To detect catalytically active proteasome subunits we used proteasome activity probe—Me4BodipyFL-Ahx3Leu3VS (UbiQbio, Amsterdam, Netherlands) according to the protocol described in (De Jong et al., 2012 (link)). Obtained lysates (app. 20 μg of total protein) were mixed with 0.5 µL of probe and incubated for 1 h at 37°C. SDS-PAGE was performed and catalytically active proteasome subunits were revealed by using ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA) (excitation wavelength 480 nm and emission wavelength 530 nm). To ensure an equal protein load the gel was incubated with the ROTI®Blue quick protein stain (Carl Roth, Karlsruhe, Germany).