Figure 1 Flow cytometric analysis of phospho-epitope levels correlates to Western blotting techniques. (A) Jurkat cells were either unstimulated or treated with PMA (50 nM), PMA (50 nM) and ionomycin (IO, 1 mM), or anisomycin (2 mg/ml) for 10 min at 37C. Cells were then divided and lysed for Western blot analysis or fixed with formaldehyde and permeabilized with methanol for flow cytometric analysis (using optimal techniques, as discussed in the text) with pERK1/2 Alexa (Ax) 488, pp38 Ax647, or pJNK Ax647. Unconjugated mAbs were used for Western blots. Unstimulated samples appear as open traces in the FACS plots. (B) U937 cells were left unstimulated or treated with IFN-g (50 ng/ml), IL-4 (10 ng/ml), or GM-CSF (10 ng/ml) for 10 min at 37C. The cells were split as above, and analyzed with pStat1 Ax488, pStat5 Ax488, and pStat6 Ax647. Note there is a strong correlation between Western blotting and flow cytometric analysis with intermediate levels of pp38 and pStat5 discernible by both methods.