Fig. 5

Fig. 5 Mib is an E3 ubiquitin ligase in vitro and enhances ubiquitination of DeltaD in Cos7 cells. (A) Western blot analysis of samples from in vitro ubiquitination assays with purified GST fusion proteins of the N-terminal half, the ankyrin repeats, or the C-terminal half containing the ankyrin repeats and ring fingers shows the self-ubiquitination activity of the ring fingers in Mib. The left half is probed with anti-HA antibodies to label ubiquitin, while the right half is probed with anti-GST antibody after stripping to label the fusion proteins. (B) Western blot analysis of the ubiquitination status of DeltaD after immunoprecipitating lysates from cells co-transfected with full-length and deletion mutants of Mib demonstrates that the ubiquitin ligase activity of Mib enhances deltaD ubiquitination. Upper panel probed with anti-FLAG to reveal ubiquitin. Lower panel was probed with anti-Delta after striping to show that IP worked. (C) Western blot analysis shows similar steady state levels of the expressed protein after co-transfection.