Figure 7.

Human CUL1 associates with ubiquitination activity in HeLa S3 lysates. (A) Anti-Polyoma beads were incubated in the presence of ^PHis6hCUL1, HeLa S3 lysates, or both as indicated, were washed five times with lysis buffer, and were mixed with the ubiquitination reaction components (Rxn mix) ^His6yUBA1, hCDC34, BUb, and ATP-regeneration system. Incorporation of BUb into proteins present in the reactions was monitored by probing with streptavidin-HRP conjugate followed by ECL detection. (B) ^PHis6hCUL1 alone or together with hSKP1 and SKP2 was produced in insect cells and was bound to anti-Polyoma beads that then were washed and incubated at 4°C in the presence or absence of crude HeLa S3 lysates to allow bead “activation”. Activated beads then were treated as in A. Dependence on the presence of ATP and BUb in the reactions was determined by omitting these components from the reaction mix (lanes 5, 8, and 9, respectively). The entire reaction mix was omitted in lane 10. Lane 1 contained reaction mix only. P-BUb_n designates a ladder of ubiquitinated proteins produced in the reaction.