Binding of SGTA to Rpn13 selectively modulates protein quality control

Leznicki, Pawel; Korac-Prlic, Jelena; Kliza, Katarzyna; Husnjak, Koraljka; Nyathi, Yvonne; Dikic, Ivan; High, Stephen

To purify the proteasomal fraction, parental HEK293T or HEK293Rpn11-HTBH cells were transfected with the indicated plasmids using GeneJuice (Merck) and then lysed 24 h post-transfection in ice-cold buffer (50 mM sodium phosphate, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 0.5% (v/v) NP-40) freshly supplemented with complete protease inhibitor cocktail (Roche), 5 mM ATP and 1 mM DTT. The resulting lysate was pre-cleared by centrifugation (20 min, 13,000×g, 4°C), and supernatants were incubated with streptavidin beads (Thermo Scientific) at 4°C overnight. The resin was washed with lysis buffer and bound proteins were eluted with SDS sample buffer and resolved by SDS-PAGE followed by western blotting. For comparison, a fraction of the total lysates, equivalent to 1% of the input used for the pull-down experiment, was analysed in parallel.