Expression of FZD5 in human monocyte-derived macrophages and lymphocytes. (A) Human macrophages were incubated in the absence or presence of M avium or M tuberculosis (MOI 3) or LPS (10 ng/mL) for 4 hours. Total RNA was isolated, reverse transcribed, and quantitative RT-PCR for detection of FZD5 mRNA expression was performed in duplicates. One representative experiment of 3 is shown. Ctrl indicates unstimulated macrophages. (B) Human lymphocytes were incubated for 4 hours in the absence or presence of anti-CD3 or anti-CD3/CD28 antibodies. Total RNA was isolated and used for quantitative RT-PCR as described. Ctrl indicates unstimulated lymphocytes; EC, cDNA from unstimulated human umbilical high venule endothelial cells (endothelial cells have been shown to express FZD5^55,67 ; serving as positive control). (C) Protein detection of FZD5 in lysates of human immune cells. 1 × 10⁶ monocytes (Mo) resp. lymphocytes (Ly) were lysed, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the proteins transferred to a nitrocellulose membrane by Western blot.⁶⁶  Recombinant human FZD5/Fc chimeric protein (50 ng) served as control (rec. FZD5). After incubation with an anti-FZD5 antibody²⁰  blots were incubated with a peroxidase-conjugated goat-antirabbit secondary antibody, and visualization was performed by enhanced chemiluminescence.