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IP-Kinase assay

XW Xujia Wu
SX Songhua Xiao
MZ Maolei Zhang
LY Lixuan Yang
JZ Jian Zhong
BL Bo Li
FL Fanying Li
XX Xin Xia
XL Xixi Li
HZ Huangkai Zhou
DL Dawei Liu
NH Nunu Huang
XY Xuesong Yang
FX Feizhe Xiao
NZ Nu Zhang
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Cells were lysed in co-IP buffer, and non-denatured CK1α and GRK2 proteins for kinase assay were obtained using Catch and Release® v2.0 Reversible Immunoprecipitation System (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. In brief, 500 μg of indicated cell lysates were incubated with anti-CK1α antibody (#sc-74582, Santa Cruz Biotechnology, Inc) or anti-GRK2 antibody (#sc-13143, Santa Cruz Biotechnology, Inc) and 10 μl of antibody capture affinity ligand in a Catch and Release v2.0 spin column. After 12 h end-over-end shaking, the column was centrifuged, washed, and then eluted with non-denaturing elution buffer. The IP-CK1α and IP-GRK2 eluates were subjected to further kinase assay using CK1α1 Kinase Enzyme System and GRK5 Kinase Enzyme System (Promega, Madison, WI, USA) respectively according to the manufacturer’s instructions. Briefly, indicated eluates were incubated with ATP/substrate Mix for 60 min at room temperature, followed by ADP detection with ADP-Glo™ Kinase Assay (Promega Madison, WI, USA).

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