Figure 4.

ATIII C247A/C430A is retained and diffuse throughout the ER. A, ATIII WT, ATIII Cys-less, and ATIII C247A/C430A were expressed in CHO cells. Cells were fixed in buffer containing 3.7% formaldehyde; permeabilized in buffer containing 0.1% Triton X-100; and stained using anti-Myc antibodies, KDEL, and giantin primary antibodies, as indicated, goat anti-mouse IgG secondary antibody, and DAPI. Cells were imaged using a confocal epifluorescence microscope at ×100 oil immersion. B, ATIII WT, ATIII Cys-less, and ATIII C247A/C430A were expressed in CHO cells. Cells were lysed in MNT. Media and lysate fractions were collected and split equally between DTT-treated and -nontreated fractions. Sucrose gradients were generated by solubilizing sucrose into MNT, and a 10–40% gradient was established. Samples were then laid on top of the sucrose gradient. Gradients were then centrifuged at 38,000 rpm for 18 h. Samples were taken from the gradient by pipetting 1 ml from the top of the gradient. Samples were TCA-precipitated and then resolved on a 9% reducing SDS-PAGE and imaged by Western blotting with Myc tag antibody.