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Figure S2. apq12Δ cells do not have an ER trafficking defect. (A) Pulse-chase analysis of carboxypeptidase Y maturation in WT, apq12Δ, and sec23-1 (positive control) cells. These experiments were performed essentially as described previously (Wilson, J.D., Y. Liu, C. Bentivoglio, and C. Barlowe. 2006. Traffic. 7:1213-1223). In brief, equal amounts of cells were harvested and washed once and resuspended with media lacking sulfate. Cultures were then incubated for 5 min at 23°C before a 5-min pulse with Promix-L [³⁵S] in vitro cell label (GE Healthcare), chased with unlabeled methionine and cysteine, and harvested at the time points indicated. The chase was terminated by the addition of 20 mM sodium azide at 4°C. Cell extracts were made by glass bead lysis. Carboxypeptidase Y was immunoprecipitated by incubating lysates with α-carboxypeptidase Y antibody and protein A-Sepharose for 2 h at room temperature. Immunoprecipitates were loaded on a 10% Tris-HCl polyacrylamide gel. The gel was dried down and exposed to a phosphorscreen and was imaged using a Storm 960 phosphoimager. (B) A FACS-based GFP UPR assay was performed as described previously (Wilson, J.D., Y. Liu, C. Bentivoglio, and C. Barlowe. 2006. Traffic. 7:1213-1223). WT, apq12Δ, and erv25Δ (positive control) were transformed with the UPRE (UPRE-GFP) reporter construct (CBB1794). Transformed colonies that were able to grow on media lacking uracil were treated with 2 mM DTT for 2.5 h before FACS analysis to ensure that cells were harboring only one copy of the plasmid. A constitutively active UPR is a hallmark of a protein trafficking defect. The increased expression of GFP in both WT and apq12Δ cells occurred only when cells were treated with DTT.