Figure 4.

Secretion and purification on sucrose density gradients of PLAP-sec and NTR-sec in transfected FRT cells. (A) Schemes of the secretory forms of PLAP (PLAP-sec) and NTR-PLAP (NTR-sec). (B) Immunoprecipitation of PLAP-sec and NTR-sec from the apical and basolateral media after overnight labeling of transfected FRT cells grown on filters. (C) PLAP-sec– and NTR-sec–enriched fractions were purified on sucrose density gradients. FRT cells expressing NTR-sec, after labeling overnight with [³⁵S]cys, were lysed and layered on a sucrose density gradient. Twelve fractions were collected from top to bottom after centrifugation to equilibrium. FRT cells expressing PLAP-sec were lysed, and after lysis they were purified on sucrose density gradients. Proteins of cells expressing PLAP-sec were TCA precipitated, run on SDS-PAGE, transferred to nitrocellulose, and blotted with an antibody against the ectodomain of PLAP. Both PLAP-sec and NTR-sec are almost exclusively restricted to the bottom fractions.