Figure 3. RB1B2 with mutant Bbox domains possessed auto-ubiquitination activity.
(i) WB image showing the results of the E3 ligase auto-ubiquitination assay of RB1B2 harboring the C142S, C145T, C195F, and the mutation of Ala130 to Ser, Thr, and Val. Lane 1 does not contain the RB1B2 protein. (ii, iii) The WB images of the bands for the activated E2∼Ub and ubiquitin are shown separately; these bands were part of the same image as (i) but were excised due to their brightness and dynamic range problems for clarity. Antibody used with all four figures was specific for biotinylated ubiquitin. (iv) WB of the stock solutions of the various His-tagged RB1B2 constructs. No His-tagged RB1B2 protein was used in lane 1, which served as control in which the E3 was omitted. This image was taken from a separate gel because the antibody was specific for the His6-tag.