FIG. 3.

Threonines 60 and 61 and proline 62 are required for SLBP degradation. (A) Mutations in the SFTTP sequence are shown at the left of each blot. The mutant SLBP constructs shown in panel A were stably transfected into HeLa cells. The cells were synchronized by a double-thymidine block, and protein extracts were prepared at 2-h intervals after release from the double-thymidine block. The levels of SLBP were analyzed by Western blotting using the anti-SLBP antibody that detects both the endogenous and His-tagged SLBP (His-SLBP). Lane 1 contains cells blocked at the G₁/S border, and lanes 2 to 7 contain cells after release from the block. The time after release is indicated above each lane. (B) Flow cytometry analysis of the cells transfected with the T60/T61 mutant of SLBP. Chromosome content is shown on the x axis, and cell number is shown on the y axis.