CCN1 expression in CD34⁺cells and in human plasma. (A) RNA from CD34⁺ cells, HUVECs and SMCs were isolated, reverse transcribed and amplified by PCR using specific primers for CCN1 and GAPDH. (B) Cell lysates from CD34⁺ cells, HUVECs and SMCs were subjected to Western blot analysis using antibodies against CCN1 and GAPDH. (C) Immunofluorescent staining of CD34⁺ cells and HUVEC with antibodies against CCN1, CD34⁺ and CCN1, von Willebrand factor (VWF), respectively. Nuclei were counterstained with Hoechst (original magnification ×1000). (D) Plasma samples from healthy donors (#1-#6) were subjected to Western blot analysis using antibodies against CCN1. Recombinant CCN1 was used as positive control (M, protein marker). (E) CCN1 standards containing 1, 10, or 100 ng/mL of recombinant CCN1 protein and plasma from healthy donors (1 and 2), untreated and preabsorbed with a neutralizing antibody against CCN1 were subjected to Western blot analysis using antibodies against CCN1. One representative blot of 3 independent experiments is shown.