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. Author manuscript; available in PMC: 2019 Mar 21.
Published in final edited form as: Methods Mol Biol. 2018;1844:277–288. doi: 10.1007/978-1-4939-8706-1_18

Table 2.

Nearly all the proteasomes can be isolated by differential centrifugation at 150,000 × g for 1 h. Ub conjugates and shuttling factors (e.g. Rad23b and Ddi2) are co-isolated from normal cells. However, in the absence of Ub conjugates (after E1 Ub-activating enzyme inhibitor MLN7243 treatment), the levels of Rad23b and Ddi2 in the proteasome-rich fraction decreased markedly. HEK293 cells were treated with 5 μM of the E1 inhibitor (MLN7243) for 1 h. Proteasome-rich fractions were prepared by the differential centrifugation method. Levels of proteins in total cell extracts and proteasome-rich fractions were analyzed by infrared Western blot. These data represent proteins recovered in the proteasome-rich fraction as a percentage of proteins from the cell extract.
Subunits and proteasome associated proteins Untreated Control After depletion of Ub conjugates
Ub conjugates 81 +− 4% Not detectable
Rpt6 (19S) 96% 98%
β5 (20S) 100 +− 0% 100 +− 0%
Rad23b 33 +− 4% 9 +− 2%
Ddi2 23 +− 4% 11%
p97 (VCP) 95 +− 5% 98%