Figure 5.

Syndecan-3 is involved in Notch processing by ADAM17/TACE. (A) Numb protein levels are increased in sdc3^−/− SCs analyzed by Western blotting and rescued to wild-type levels by exposure to low oxygen. Ponceau staining is shown as a loading control. (B) Explanted wild-type and sdc3^−/− SCs were transfected with N^FL-6mt, ΔE-6mt, or IC_V-6mt in addition to either empty vector or CSL-Luc and CMV-LacZ as a transfection efficiency control. Reporter activity analyzed 24 h after transfection revealed that ΔE-6mt and IC_V-6mt, but not N^FL-6mt, ectopic expression rescues Notch reporter activity in sdc3^−/− SCs to wild-type levels. (C) Wild-type and sdc3^−/− SCs were isolated, cultured, and transfected as in B. 24 h later, they were analyzed by Western blotting to detect the myc-tag epitope. Ectopic Notch1 accumulates in sdc3^−/− cells transfected with N^FL-6mt (N^TM), but not in sdc3^−/− cells transfected with either ΔE-6mt or IC_V-6mt (n = 3). The same blot was stripped and reprobed to detect NICD generated by γ-secretase cleavage. Although NICD was detected in samples transfected with IC_V-6mt, no NICD was detected upon N^FL-6mt or ΔE-6mt ectopic expression (n = 2). β-galactosidase is shown as a loading control. (D) Explanted wild-type and sdc3^−/− SCs transfected with N^FL-6mt were treated as indicated 24 h after transfection. Western blots of cell lysates were probed for the myc-tag epitope and β-galactosidase. N^TM band intensities were quantified and values were normalized to the corresponding β-galactosidase band intensities plotted, with the normalized value corresponding to wild-type nontreated cells set at 1. Open bars, wild type; shaded bars, sdc3^−/−. Error bars indicate SEM. *, P < 0.05; #, P > 0.05. Molecular mass standards are indicated next to gel blots (kD).