Calyculin A is a serine/threonine phosphatase inhibitor that inhibits the activity of protein phosphatases PP1 and PP2A. Human carcinoma A431 cells treated with calyculin A for 30 minutes can undergo significant threonine phosphorylation, as shown by western blotting using anti-Phospho-Akt (Thr-34), as compared to untreated, control cell lysates. Confluent cultures of A431 cells were serum starved overnight. Cells were then either left untreated or treated with Calyculin A at a final concentration of 10nM for 30 minutes at 37°C. Cells were lysed in 1% SDS, 1.0mM sodium ortho-vanadate, 10mM Tris (pH 7.4) buffer. Protein concentration was determined using the BCA method (Pierce) before diluting to final concentration and buffer.