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. 2000 Feb 25;97(6):2497–2502. doi: 10.1073/pnas.060025497

Figure 2.

Figure 2

Overexpression of Rpn1p inhibits degradation of N-end rule substrates. A UPR-based pulse–chase assay was used (see Results). S. cerevisiae JD52 (UBR1) cells coexpressing either DHFR-HA-UbR48-Leu-βgal (A) or DHFR-HA-UbR48-His-βgal (B), and either Rpn1p (expressed from its own promoter in pRS425) or pRS425 alone were labeled with [35S]methionine for 5 min at 30°C, followed by a chase for 0, 10, and 30 min. Cell extracts were immunoprecipitated with both anti-βgal and anti-HA antibodies, followed by SDS/12% PAGE, autoradiography, and quantitation using PhosphorImager (Molecular Dynamics). (C) His-βgal and Leu-βgal decay curves calculated from the UPR-based data in A and B as described (28, 32). The bands of X-βgals and the reference protein DHFR-HA-UbR48 (“ref”) are indicated.