(A) Analysis of PSer¹³³-CREB and α_IIbβ3 by double (anti-PSer¹³³-CREB polyclonal rabbit serum + anti-CD41a MoAb followed by GAR-FITC + GAM-TRIC) immunofluorescence in primary CD34⁺hematopoietic progenitor cells stimulated for various days with 100 ng/mL of TPO. CD34⁺ cells were analyzed immediately after purification (day 0) and after 3 or 6 days of liquid culture in the presence of 100 ng/mL of TPO by either phase contrast or fluorescence microscopy. Green nuclear fluorescence corresponds to PSer¹³³-CREB positivity, red membrane and cytoplasmic fluorescence corresponds to CD41a positivity, and blue fluorescence corresponds to nuclear counterstaining with DAPI. Results from one representative experiment of three separate experiments are shown. (B) Western blot analysis of PSer¹³³-CREB and whole CREB protein performed in CD34⁺ hematopoietic progenitor cells immediately after purification (lane 1) or after 6 days of liquid culture in the presence of 100 ng/mL of TPO (lane 2). The positive control is represented by HEL cells (lane 3) treated with TPO for 30 minutes. The densitometric analysis is expressed in arbitrary units (a.u.).