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. 1998 Aug 10;142(3):651–663. doi: 10.1083/jcb.142.3.651

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Mislocalization of Vti1p in vac7-1 cells. (A) Wild-type (NBY86; pho8Δ-X pep4Δ-X) and vac7-1 (NBY85; vac7-1 pho8Δ-X pep4Δ-X) cells harboring pSN92 (ALP) were prepared for double labeling indirect immunofluorescence using the α-ALP mAb 1D3-A10 and affinity-purified antibodies against Vti1p, as described in Materials and Methods. (B) Pep12p was immunolocalized in wild-type (NBY86; pho8Δ-X pep4Δ-X) and vac7-1 (NBY85; vac7-1 pho8Δ-X pep4Δ-X) cells harboring pSN92 (ALP) as well as in cells that do not synthesize Pep12p (vpt13), as described in Materials and Methods. In both cases, cells were also visualized using DIC microscopy. (C) Kinetics of processing of ALP and Vps10p-Δ10* in vac7-1 cells. SEY6210 (wild-type) and LWY2809 (vac7-1) PEP4 cells were labeled with [³⁵S]Met for 10 min and chased by adding unlabeled methionine and cysteine each to a final concentration of 50 μg/ml. At the indicated times proteins were immunoprecipitated from cell extracts using polyclonal antibodies against ALP and Vps10p. The resulting immunoprecipitates were subjected to SDS-PAGE and fluorography. The products of PEP4-dependent proteolysis of both ALP and Vps10p-Δ10* are indicated using asterisks.

Mislocalization of Vti1p in vac7-1 cells. (A) Wild-type (NBY86; pho8Δ-X pep4Δ-X) and vac7-1 (NBY85; vac7-1 pho8Δ-X pep4Δ-X) cells harboring pSN92 (ALP) were prepared for double labeling indirect immunofluorescence using the α-ALP mAb 1D3-A10 and affinity-purified antibodies against Vti1p, as described in Materials and Methods. (B) Pep12p was immunolocalized in wild-type (NBY86; pho8Δ-X pep4Δ-X) and vac7-1 (NBY85; vac7-1 pho8Δ-X pep4Δ-X) cells harboring pSN92 (ALP) as well as in cells that do not synthesize Pep12p (vpt13), as described in Materials and Methods. In both cases, cells were also visualized using DIC microscopy. (C) Kinetics of processing of ALP and Vps10p-Δ10* in vac7-1 cells. SEY6210 (wild-type) and LWY2809 (vac7-1) PEP4 cells were labeled with [³⁵S]Met for 10 min and chased by adding unlabeled methionine and cysteine each to a final concentration of 50 μg/ml. At the indicated times proteins were immunoprecipitated from cell extracts using polyclonal antibodies against ALP and Vps10p. The resulting immunoprecipitates were subjected to SDS-PAGE and fluorography. The products of PEP4-dependent proteolysis of both ALP and Vps10p-Δ10* are indicated using asterisks.