Akap9

Cells were washed twice with cold PBS and lysed in RIPA buffer (1× PBS, 1% NP40, 0.1% SDS, 5 mM EDTA, 0.5% sodium deoxycholate, and 1 mM sodium orthovanadate) containing protease inhibitors. Whole protein extracts were resolved on a 10% SDS polyacrylamide gel and electrotransferred to polyvinylidene fluoride membranes (Millipore, USA), which were then blocked in 5% non-fat dry milk in Tris-buffered saline (pH 7.5; 100 mM NaCl, 50 mM Tris, and 0.1% Tween-20) and immunoblotted with AKAP9 (Santa Cruz, USA) at 4°C overnight followed by HRP (horseradish peroxidase)-labeled secondary antibody (Santa Cruz) and detected by chemiluminescence. α-Tubulin expression was used as a protein-loading control. The intensity of protein bands was quantified with the Quantity One software (4.5.0 basic, Bio-Rad).

Cells were washed twice with cold PBS and lysed in RIPA buffer (1× PBS, 1% NP40, 0.1% SDS, 5 mM EDTA, 0.5% sodium deoxycholate, and 1 mM sodium orthovanadate) containing protease inhibitors at 4 °C followed by vortex and centrifugation at 14,000 rpm at 4 °C for 10 min. Total proteins (500 ug/sample) were pre-cleaned with 40 μl A-G beads (Santa Cruz) before immunoprecipitation with 3 μg control IgG (Santa Cruz Biotechnology), AKAP-9, or CIP4 antibody at 4°C overnight. After incubation with 40 μl A-G beads at 4°C for 6 hours, the immunoprecipitates were washed with PBS containing 0.2% NP-40 for 5 times. The immunoprecipiated protein complexes were then released by boiling in 2×SDS-PAGE sample buffer for 5 minutes and used for immunoblotting with both AKAP-9 (Abcam) and CIP4 antibodies (Santa Cruz).