Downregulation of FAP-1 expression in TEC in the presence of cycloheximide. TEC on day 4 of primary culture were incubated for 24 hours with 10 μg/mL cycloheximide (CHX) or in medium (M). (A) mRNA was extracted and submitted to RT-PCR. In two independent experiments, treatment of TEC with cyloheximide induced a clear decrease in FAP-1 mRNA levels, whereas β-actin expression was not modified. (B) Western blot analysis of FAP-1 expression. TEC were solubilized, and 20 μg total protein in each condition was analyzed on SDS-PAGE 7.5% and transferred to PVDF membranes. The blot was incubated with anti–FAP-1 antibody and then with peroxidase-conjugated antigoat Ig. Immunoreactivity was determined using the ECL chemiluminescence reaction. A major protein band (apparent molecular weight, 200 kD) was decreased in the presence of cycloheximide.