Figure 3F shows the levels of phosphorylated proteins Akt, 4E-EP1, S6, Src and Smad2/3 in mice hemopoietic stem cells expressing or lacking GRP78 receptor. More specifically, mice HSCs expressing GRP78 receptor (GRP78+) or HSCs that lack the receptor (GRP78-) were treated with or without growth factor rmCripto for 15 min, 90 min, 12 hr, and 36 hr. Then, cells were stained with anti-GRP78 and fixed in BD Cytofix/CytopermTM Fixation and Permeabilization Solution. Next, fixed cells were stained with Alexa Fluor 488 conjugated antibodies specific to phosphorylated Akt,4E-EP1, S6, Src, and Smad2/3. Finally, the fluorescence representing the level of proteins phosphorylation was estimated using flow cytometry. As a result, each graph represents the change of mean fluorescent intensity (MFI) of certain phosphorylated protein in GRP78+ or GRP78- cells treated or non-treated with Cripto. Each point represents MFI +/- SD of four experiments. Overall, the figure shows that Cripto-treated GRP78+ HSCs demonstrate a significant increase in phosphorylated Akt, 4E-BP1 and S6 comparing to non-treated cells (red lines and points marked with asterisks at 15 min and 90 min of treatment). In addition, GRP78- HSCs showed no response to Cripto as the MFI is similar between Cripto-treated or untreated GRP78- cells at all time points. Therefore, the data suggest that Cripto signaling in hemopoietic stem cells is associated with Akt signaling pathway.