Research Article

The Laminin-α1 Chain-Derived Peptide, AG73, Binds to Syndecans on MDA-231 Breast Cancer Cells and Alters Filopodium Formation

Figure 2

Sdcs 1-4 as well as glypican (Gpc)1 are expressed in MDA-231 cells. (a) qPCR was used to detect the message levels of each Sdc and glypican 1 (Gpc1) in breast cancer cell lines. Bars: mean Ct values are normalized to . All reactions were run in triplicate and repeated three times, and gene expression was normalized to the housekeeping gene GAPDH. (b) Sdcs 1 and 4 bind to AG73. For this solid-phase assay, wells were uncoated (0) or coated with AG73. Cells were pretreated with either heparin (H) or trypsin (T) before they were lysed. Controls were left untreated (0). The MDA-231 cell lysate binds to AG73 but not to the scrambled peptide (data not shown). The wells were then incubated with antibodies to Sdcs and detected using a secondary-HRP conjugate. Heparin and trypsin pretreatment inhibited Sdcs 1 and 4 binding to AG73. Bars: mean OD . (c) AG73 binds a >250, 90, and 50 kDa biotinylated protein from the surface of MDA-231 cells. Biotinylated MDA-231 cell membrane was incubated with AG73 (A) or scrambled peptide (S) as in (b). Proteoglycan bands usually appear as a smear due to the heparan sulfate and chondroitin sulfate glycosaminoglycan chains.
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