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. 1997 May 5;137(3):779–791. doi: 10.1083/jcb.137.3.779

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Phosphorylation state of His-pro-uPA^wt and His-prouPA^138E/303E vs untagged pro-uPA^wt in A431 stable clones. Subconfluent A431 cells, stably overexpressing His-pro-uPA^wt (A) or His-pro-uPA^138E/303E (B), have been metabolically labeled with either [³⁵S]methionine or with [³²P]phosphate for 18 h. The resulting conditioned medium was subjected to Ni-NTA chromatography to recover the histidine-tagged pro-uPAs. The Ni-NTA excluded proteins were incubated with 5B4 anti-uPA antibody to isolate untagged pro-uPA^wt. Each sample, deriving from 0.5 × 10⁶ cells, has been analyzed onto a 12.5% SDS-PAGE under reducing conditions.