Figure 6.

Immunoprecipitation (I.P.) and Western Blot analysis of culture medium shows that paracrine factors are secreted from hgPSC-derived cardiac colonies. A) Silver stained SDS-PAGE gel detected IGF-1 by IP after 12hrs of culture, increasing in intensity through 48hrs of culture. B). Silver staining shows TGFb secretion within 12hrs becoming more intense after 48hrs. C) VEGF is detectable by about 24hrs of culture. D) Western analysis of CTGF secretion is detected within 24hrs while NRG-1 secretion (E-F) is detected after 48hrs of culture. Cardiac differentiation of hgPSCs from two patients are shown for Nrg1. All IP experiments were run with a lane containing IgG alone to identify the heavy and light chain bands. 2μl from all samples were analyzed using a nano-drop ND-8000 (Thermo Fisher Inc.) to normalize protein concentrations. G) ELISA bar graph data shows quantitative amounts of TGFü secretion detected within 12-48 hours of culture. ELISA data confirm the silver stained SDS PAGE gels, which detected a similar secretion pattern as found in the ELISA.