FIGURE 4.

Incorporation of α4s does not alter the catalytic activities of the proteasome. A, extracts of HEK293T cells expressing both α4s-FLAG and α4-shRNA (α4s-FLAG cells) were fractionated by glycerol gradient centrifugation and subjected to immunoblotting with the indicated antibodies. B, lysates of α4s-FLAG cells, α4*-FLAG cells, and mock HEK293T cells were fractionated by glycerol gradient centrifugation, and the peptidase activities of each fraction were measured in the absence (left) and presence (right) of 0.025% SDS. Arrows indicate the peak locations of 20 S CPs and 26 S proteasomes. C, fractions 15 and 25 in B were subjected to immunoblotting for the indicated antibodies. Data are representative of three independent experiments (A–C). D, cell extracts of these three cell lines were assayed for ATP-dependent protein degradation activity using ³⁵S-labeled ornithine decarboxylase as a substrate. Mean ± S.D. (n = 3).