FIG. 6.
A six-Myc epitope tag at the N terminus stabilizes cyclin G11-137. (A) NIH 3T3 cells were transfected with six-Myc-tagged cyclin G11-137 or untagged cyclin G11-137 for 24 h, followed by treatment with cycloheximide (0.1 μg/ml). Cells were lysed at the indicated times posttreatment, and lysates were separated by 12% SDS-PAGE followed by immunoblotting with cyclin G1 and actin antibodies as described above. Cyclin G1 protein was quantitated using an Odyssey system. (B) NIH 3T3 cells were transfected with constructs expressing six-Myc-tagged and untagged cyclin G11-137, with or without HA-ubiquitin, for 24 h and then either left untreated or treated with 25 μM MG132 in dimethyl sulfoxide for 5 h. Cell lysates were separated by 12% SDS-PAGE and immunoblotted with anti-cyclin G1, antiactin, or anti-GFP antibodies, as indicated on the right. On the left are indicated one or two molecules of untagged endogenous ubiquitin (Ub1 and Ub2) or ectopic HA-tagged ubiquitin (HA-Ub1 and HA-Ub2) conjugated to cyclin G1. Long and short exposures of the immunoblot of cyclin G1 are indicated.