Fig. 3.
CPL1 dephosphorylates intact AtCTD. GST-AtCTD protein was phosphorylated by activated mitogen-activated protein kinase ERK2. After overnight dialysis, GST-AtCTDO was incubated with 0.1 μg (1×) or 0.2 μg (2×) of the GST-CPL11-638 preparation (CPL1) or calf intestine alkaline phosphatase (CIAP) in reaction buffer (50 mM Tris, pH 5.5 or 7.0) without or with 10 mM MgCl2 for 3 h at 37°C. Phosphorylation status was gauged after resolving the products by SDS/PAGE by immunoblotting with mAbs H14 and H5 that detect Ser-5-PO4 and Ser-2-PO4, respectively. (Lower) The polypeptide compositions were visualized by staining with Coomassie brilliant blue dye (CBB). The amount of GST-AtCTD applied to the gels was 0.1 μg for H14 and 1 μg for H5 and CBB staining.