Cytokine antibody arrays show decreased IL-4 expression in Fgfr-1^−/− embryoid bodies. (A) Filters spotted in duplicate with antibodies against 62 different cytokines were incubated with conditioned medium from Fgfr-1^+/− or Fgfr-1^−/− embryoid bodies. Immunoreactivity was visualized by ECL. Positive control protein spots are indicated by white boxes in the upper left corner of each filter. Specific changes in antibody reactivity are boxed (marked 1-4). (B) Enlargement of marked filter areas (1-4) in panel A, showing spots with reduced or increased cytokine levels in conditioned media (CM^−/− compared with CM^+/−). (C) Quantification of average pixel intensity of duplicate spots for each cytokine, IL-4, SCF, TIMP-1, and CTACK, in CM^−/− and CM^+/−. (D) Real-time PCR analysis of candidate cytokines in Fgfr-1^+/− and Fgfr-1^−/− embryoid bodies at 4 days of differentiation. (E) Induced vessel formation shown by VEGFR-2 immunostaining (red) in Fgfr-1^−/− embryoid bodies as a result of treatment with neutralizing anti–IL-4 antibodies (5 μg/mL) from day 0 to day 4 compared with control isotype-matched IgG treatment. Bars represent 100 μm. (F) Quantification of Fgfr-1^+/− embryoid body vascularization in response to treatment with control IgG or anti–IL-4 antibody. (G) Higher magnification of vessels treated with IgG or anti–IL-4 antibody. Bars represent 50 μm.