Anti grk2 antibody

Crude cell extracts were prepared by lysing cells using RIPA lysis buffer (Pierce) supplemented with phosphatase and protease inhibitor cocktails (Roche). The extracted proteins (20–50 μg) were resolved with 10% SDS polyacrylamide gels and transferred onto a nitrocellulose membrane (Millipore) according to standard protocols. Polyclonal anti-CXCR4 antibody (1:1000, Abcam), anti-c-kit antibody (1:500, Abcam), anti-CXCR4-phospho-serine 339 antibody (1:500, Abcam), anti-ERK1/2 antibody (1:1000, Cell Signalling Technology), anti-phospho-ERk1/2 antibody (1:1000, Cell Signalling Technology), anti-p38 antibody (1:1000, Cell Signalling Technology), anti-phospho-p38 antibody (1:1000, Cell Signalling Technology), anti-GRK6 antibody (1:1000, Santa Cruz), and anti-GRK2 antibody (1:1000, Santa Cruz) were used overnight at 4 °C. This was followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1:10,000; Pierce). Peroxide activity was detected using the enhanced chemiluminescence Supersignal West Dura system (Pierce). As a loading control, mouse anti-beta-actin antibody was used at a concentration of 1:1,000.

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).