Pull down assays using amylose resin

Proteins were diluted to 3 μM in 40 μL buffer HST300, unless otherwise noted, and mixed with 20 μL amylose resin (NEB) equilibrated with buffer HST300 or buffer with reduced NaCl concentration of 100 mM. One-third of this mixture was taken as input fraction and the rest two-thirds were incubated at 4°C for 30 min. Amylose-bound proteins were separated from unbound fraction by spinning down the amylose resin and washing with 500 μL buffer HST300 four times. The input and bound fractions were analyzed by Tricine–SDS-PAGE using normal gels and Phos-tag gels. The gels were stained with Coomassie brilliant blue (CBB). Phos-tag acrylamide gels containing 50 μM Phos-tag AAL-107 (NARD institute) were prepared according to the manufacture’s protocol and were used to detect the mobility shift caused by phosphorylation.