FIG. 5.

ARIP3 interacts with other sumoylated proteins. COS-1 cells were transfected with 1 μg of empty pFLAG-CMV2 or pFLAG-ARIP3 and 1.5 μg of empty pSG5 or pSG5-His-SUMO-1. Forty-eight hours after transfection, cells were collected and lysed either in RIPA-1 buffer containing 10 mM NEM or in a denaturing buffer containing 2% SDS in 10 mM Tris-HCl (pH 8.0)-150 mM NaCl. Samples lysed in the latter buffer were heated at 95°C for 10 min. Before immunoprecipitation (IP), these samples were diluted (1:10) with a mixture of 10 mM Tris-HCl (pH 8.0) and 150 mM NaCl containing 1% Triton X-100. Five percent of the cell extracts were immunoblotted with the anti-FLAG antibody (A), and the rest of the samples were immunoprecipitated with the anti-FLAG antibody followed by immunoblotting with the anti-SUMO-1 antibody (B) or the anti-FLAG antibody (C). WB, Western blotting.