Anti tnfaip8

For Western blot analysis, cells were lysed for 20 min at 4°C in 1% Triton X-100 lysis buffer containing protease and phosphatase inhibitors. Cell debris was removed by centrifugation at 14,000 × g for 20 min at 4°C. For subcellular fractionation, membrane and cytoplasmic proteins were separated using Qproteome Cell Compartment Kit (QIAGEN) according to the manufacturer’s instructions. The protein concentration of the lysates was determined by Bradford assay. Equal amounts of total protein were resolved by SDS-PAGE, transferred to membranes, immunoblotted with specific primary and secondary antibodies, and the signals were detected by chemiluminescence (Pierce). Primary antibodies against p-AKT(S473), AKT, Myc, as well as anti-Myc conjugated to HRP, were purchased from Cell Signaling Technology and used according to the manufacturer’s instructions. Anti-TNFAIP8 (1:500) was purchased from ProteinTech Group and Anti-RAC1 (1:500) was purchased from Millipore. Anti-Flag (1:2000), Anti-Flag-M2-HRP, anti-β-actin (1:3000) and anti-GAPDH antibody (1:3000) were purchased from Sigma. HRP-conjugated secondary anti-mouse and anti-rabbit IgG (1:1500) were purchased from GE Healthcare. The densitometric quantification of Western blot signals was performed using ImageJ software. Paired t-test was used to evaluate the statistical significance of the results.