Figure 2.

Cellularly retained ATIII C247A/C430A is inactive and contains a higher level of free thiols. A, WT ATIII and ATIII disulfide mutants were expressed in CHO cells. Cells were radiolabeled with [³⁵S]Cys/Met for 30 min and chased for the indicated times. At each time point, cell lysate and media were collected and processed as described in the legend to Fig. 1A. B, quantification of ATIII secretion from A. The lysate and media were quantified, and ATIII secretion is presented as a percentage of ATIII in the media to ATIII in the 10-min lysate. C, ATIII and ATIII disulfide mutants were expressed in CHO cells, radiolabeled with [³⁵S]Cys/Met for 30 min, and chased for 3 h. Cells were lysed in MNT buffer. Cell lysate and media were collected, and ATIII was immunoprecipitated using anti-Myc antibody and washed with 0.5% CHAPS buffer. ATIII was then eluted from the immunoprecipitation beads by incubation with 0.5 mg/ml c-Myc peptide for 1 h at 37 °C. Samples were then evenly split between treated and nontreated samples. 0.648 mg (2 units) of thrombin were added to treated samples, whereas nontreated samples received an equal volume of water. Samples were then incubated at 37 °C for 1 h and resolved by reducing 9% SDS-PAGE. D, WT ATIII and ATIII disulfide mutants were expressed in CHO cells, radiolabeled with [³⁵S]Cys/Met for 30 min, and chased for 30 min. Cells were lysed in MNT buffer. To treated samples, PEG-maleimide was added to a final concentration of 1.4 mm, and to the nontreated samples, N-ethyl maleimide was added to a final concentration of 5 mm. Samples were incubated at room temperature for 30 min. Treated samples were then quenched with DTT at a final concentration of 100 mm. ATIII was immunoprecipitated using anti-Myc antibodies, and samples were resolved by reducing 9% SDS-PAGE. E, quantification of PEG-maleimide–unmodified ATIII from D. The percentage PEG-maleimide–unmodified ATIII was determined by quantifying the amount of unmodified ATIII in PEG-maleimide–treated and –nontreated samples and dividing the treated sample by the untreated sample. All experiments are representative of three independent experiments. Error bars, S.D.