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Fig. 4. Expression, secretion and localization of pro-uPA to caveolae are downregulated in AS-cav-1 HCT 116 cells. (A) Conditioned media from control and AS-cav-1 HCT 116 cells were collected and the cells were solubilized in lysis buffer containing 1% Triton X-100, 60 mM octylglucoside. Media and cell lysates (25 µg protein) were analysed by SDS-PAGE and immunoblotted with anti-human-uPA antibodies. Immunoblots are shown in triplicate and are representative of at least three experiments. (B) Equal amounts of protein for control and AS-cav-1 HCT 116 cells were subjected to subcellular fractionation on a sucrose gradient after homogenization in sodium-carbonate buffer, pH 11.0. Fractions were collected from the top of the gradient and equal-volume aliquots of fractions 2-11 were analysed by SDS-PAGE and immunoblotted with anti-human-uPA antibodies. Immunoblots are representative of at least three experiments.
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