Description
Calyculin A is a serine/threonine phosphatase inhibitor that inhibits the activity of protein phosphatases PP1 and PP2A. Cells treated with calyculin A for 30 minutes can undergo significant threonine phosphorylation, as shown by westernblotting using anti-Phosphoserine/threonine, as compared to untreated, control cell lysates.Fibroblast from rabbit spleen were harvested to generate a primary fibroblast cell line. Confluent cultures of rabbitfibroblasts were serum starved for 2 hours. Cells were then either left untreated or treated with Calyculin A(100nM) for 30 minutes at 37 degree C. Cells were lysed in 1% SDS, 1.0mM sodium ortho-vanadate, 1mM sodium fluoride in 10mM Tris (pH7.4) buffer. Protein concentration was determined using the BCA method (Pierce) before diluting to final concentration and buffer