Odyssey ir software version 1

In general, cells were harvested 48 h after DNA transfection or 120 h after siRNA transfection or DOX-induced SAE2 knockdown. After washing twice with 1× PBS, the cells were directly lysed in 2× SDS sample buffer (4% SDS, 20% glycerol, 0.004% bromophenol blue, and 0.125 M Tris–HCl pH ∼6.8). Samples were sonicated for 3 × 10 s to shear DNA and quantitated using BCA protein assay kit. 2 M β-mercaptoethanol was added to the protein samples, which were then boiled at 95°C for 5 min, before separation using SDS-PAGE, and Western blotting. Western blot results were visualized using the Odyssey IR imager (LI-COR) that can detect both IRDye 680- and 800-conjugated secondary antibodies (1:10 000). Quantification of Western blots was performed using Odyssey IR software, version 1.2 (LI-COR). For Akt immunoprecipitation and Western blot, anti-Akt antibody (40D4, mouse mAb; Cell Signaling) was first used for immunoprecipitation. Anti-SUMO1 (C9H1; rabbit mAb; Cell Signaling) and Clean-Blot IP Detection Kit (Thermo Scientific) were used for Western blot.