Ab166628

To prepare cell lysates, the pellets were resuspended in lysis buffer (20 mM Na-phosphate pH 6.8, 10 mM DTT, 1mM EDTA, 0.1% Tween 20, 1 mM PMSF, and EDTA-free protease inhibitor mini tablets (Thermo Fisher, #A32955)) and rotated at 4°C for 30 min. Cells were sonicated in a 4°C water bath-based sonicator (Bioruptor: 8 times at level 4.5 and 50% duty cycle) and centrifuged for 20 min at 200g at 4°C. The concentration of protein in the supernatant was calculated using the Bradford reagent (Thermo Fisher, #23236) and adjusted to a same concentration for all samples. Protein aggregates were pelleted at 16,000g for 20 min at 4°C. After removing supernatants, protein aggregates were washed 2 times with NP-40 buffer (20 mM Na-phosphate pH 6.8, 2% NP-40, 1mM PMSF, and EDTA-free protease inhibitor mini tablets), sonicated 6 times at level 4.5 and 50% duty cycle, and centrifuged at 16,000g for 20 min at 4°C. Aggregated proteins were washed in wash buffer (20 mM Na-phosphate pH 6.8, 1mM PMSF, and EDTA-free protease inhibitor mini tablets), sonicated 4 times at level 3 and 50% duty cycle, and boiled in 2X SDS sample buffer. Resuspended aggregated proteins were analyzed by western blotting with antibodies directed against CK2β (Abcam ab76025) and PSMB2 (Abcam ab166628), or processed for mass spectrometry as described below.