Western analysis of STAT3, tyrosine-phosphorylated STAT3, and serine-phosphorylated STAT3.
Total (A) STAT3, (B) tyrosine-phosphorylated STAT3 (STAT3 pTyr 705), and (D) serine-phosphorylated STAT3 (STAT3α pSer 727) protein as a function of time and culture condition were quantified based on equivalent amounts of total protein by analyzing the immunoblots shown in Figure 4A-C (culture conditions 1-4), respectively, with ImageQuant software. The signal intensities for each isoform within a lane were added to yield the total amount of STAT3, STAT3 pTyr 705, or STAT3 pSer 727 protein and expressed relative to the day 0 value. Anti-STAT3 pSer 727 antibody specifically recognized the phosphorylated serine 727 site found only in the STAT3α isoform. (C) STAT3 pTyr 705 was normalized to total STAT3 levels, or (E) STAT3 pSer 727 protein was normalized to total STAT3α levels at each time point to show changes in the fraction of STAT3 that is either tyrosine- or serine-phosphorylated, respectively, during differentiation. ▴ indicates 5% O2, pH 7.25, +IL-3; ▵ indicates 20% O2, pH 7.25, +IL-3; ● indicates 5% O2, pH 7.4, +IL-3; and ● indicates 5% O2, pH 7.25, −IL-3.
