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. Author manuscript; available in PMC: 2013 Nov 2.
Published in final edited form as: Biochem Biophys Res Commun. 2012 Oct 5;427(4):768–773. doi: 10.1016/j.bbrc.2012.09.136

Figure 3. Y345H mutant Sec61 preserves translocation.

Figure 3

(A) 5 ODs of yeast transformed with CPY*-FLAG in log phase growth were labeled with 50 Ci of 35S-cysteine/methionine, lysed, immunoprecipitated with anti-FLAG antibody and fractionated by SDS-PAGE. The upper band represents fully glycosylated and translocated CPY*; the lower band represents an unglycosylated and untranslocated form. (B) Yeast transformed with DPY*-FLAG were treated as in A. SEC61 yeast treated with tunicamycin, which blocks N-linked glycosylation serves as a control for unglycosylated DPY*. For analysis of CPY* degradation (C) yeast expressing the indicated Sec61 allele and transformed with CPY*-FLAG growing in log phase were harvested, labeled with 35S-cysteine/methionine and chased with cold media for the indicated time points. Yeast were then lysed, immunoprecipitated with anti-FLAG antibody and fractionated by SDS-PAGE. (D) Quantitation of three independent experiments. Shown are mean ± SD *= p<0.05 by Student’s t-test.