Figure 6.

Syndecan-3 and Notch are in a complex on the SC membrane. (A) Syndecan-3 and Notch1 (N^FL-6mt) were coimmunoprecipitated from MM14 cell extracts with an anti–Syndecan-3 antibody 24 h after transfection, and Western blots were probed for the myc-tag epitope and for Syndecan-3 using a terminal heparan sulfate attachment antibody (Δ-HS). (B) Extracts from MM14 cells cultured in the presence or absence of an anti-Notch1 antibody, transfected as in E, and treated with or without 200 nM TIMP-3 were immunoprecipitated with an anti–Syndecan-3 antibody. Blots probed for the myc-tag epitope, then stripped and reprobed for Δ-HS. The relative levels of N^TM were quantified and plotted with the level of N^TM in the untreated sample set to 1. Molecular mass standards are indicated next to gel blots (kD). (C–F) Endogenous Notch and Syndecan-3 are present in a protein complex on the SC surface, as revealed using DuoLink with an extracellular anti-Notch1 antibody and an intracellular Syndecan-3 antibody. Two distinct Notch1 antibodies from different species show as positive complexes in Syndecan-4 (sdc4)⁺ cells (green) after DuoLink labeling (C, red), whereas elimination of the primary anti-Notch1 and anti–Syndecan-3 antibodies yielded no positive complexes (D). Endogenous Notch1 and Syndecan-3 appear as complexes (red dots) in wild-type Syndecan-4⁺ cells (E) but are absent or at background levels in sdc3^−/− cells (E). Arrows indicate which cells are magnified in the inset panels. (F) Quantification of C–E, where the mean number of DuoLink⁺ complexes/cell is plotted. n = 3. Open bars, wild type; shaded bars, sdc3^−/−. Error bars indicate SEM. **, P < 0.01; #, P > 0.05. Bars, 10 µm.