Preformed intracellular CD40L does not contribute to surface expression. (A) PBMCs were left unstimulated or TCR stimulated for 6 hours as indicated. After 2 hours of stimulation, BFA was added to the samples as indicated. Cells were stained for surface CD40L or for intracellular CD40L after fixation and permeabilization. (B) PBMCs were plated in the absence (Control) or presence of a translational inhibitor (CHX). Control samples were harvested and stained for CD40L at the indicated times (solid line). CHX was added to the treated cultures at 0, 1, 2, or 3 hours, and the CHX-treated samples were harvested and stained for CD40L at 6 hours (hatched columns; undetectable in 0 and 1 hour samples). CD40L expression is plotted relative to the 6-hour control (100%).