tBid-N is ubiquitinated in an unconventional manner. (A) HeLa cells were transfected to express HA tBid-N alone or in combination with FLAG-tagged ubiquitin. HA tBid-N was immunoprecipitated and probed with an anti-HA mAb to detect Bid and probed with anti-FLAG mAb to detect ubiquitin. Asterisks indicate heavy and light chains of anti-HA mAb. (B) MCF-7 cells expressing Myc Bid GFP were stimulated with TNF-α in the presence of MG132 for 8 h or not stimulated (control). Lysates were immunoprecipitated with an anti-GFP antibody and immunoprecipitated sequentially (seq) with an anti-Myc mAb, and immunoprecipitates (IP) were probed with an anti-Bid antibody and a P4D1 anti-ubiquitin mAb. (C) Assay to determine whether the N terminus is ubiquitinated. In the diagram, the tBid-N TAP proteins are assessed for ubiquitination. Cal–TEV–Prot A indicates the TAP tag with calmodulin- and IgG-binding sites separated by a cleavage site for TEV protease (arrows). HeLa cells were transfected to express HA-ubiquitin and TAP-tagged tBid-N. TAP-tagged tBid-N proteins were isolated with IgG beads, digested with TEV protease, and reisolated with calmodulin beads. Isolates were probed for ubiquitin with an anti-HA mAb and for tBid-N with an anti-Bid antibody. Note that in I, the Prot A sequence is still attached to the tBid-N TAP species and the anti-Bid antibody binds to it. Molecular masses (kD) of marker proteins are indicated.