Identification of a TFPI-syndecan-3 protein complex by native PAGE and immunoprecipitation.

<p>Protein lysates were isolated from cells, subjected to native PAGE analysis or immunoprecipitation experiments and analysed by Western blotting. A) Native PAGE of lysates from Sum102 cells (20 μg), HCAECs (12 μg) and HCASMCs (18 μg) immunoblotted with anti-TFPIα (top) and anti-syndecan-3 (bottom) (one representative membrane out of three is shown), B) SDS-PAGE of full length recombinant TFPIα protein immunoblotted with anti-TFPIα, and C) SDS-PAGE of syndecan-3 293T control lysate immunoblotted with anti- syndecan-3. D) HCAEC (40 μg), E) SUM102 (160 μg) and F) HCASMC (40 μg) lysates were subjected to immunoprecipitation using two different syndecan-3 antibodies (n = 2 for sc-30883, n = 1 for sc-9495) or control goat IgG (n = 2). Lysates and immunoprecipitates were analysed for the presence of endogenous syndecan-3 and TFPI by immunoblotting. Recombinant TFPIα protein (upper left panel) and cell lysate (lower left panel) was used as positive control for the immunoblotting in D-F.