Analysis of 4EBP1 (pT69) in human peripheral blood monocytes. Human peripheral blood mononuclear cells (PBMC) were either treated with 100 μM LY294002 (Sigma, Cat. No. L-9908) for 1 hour at 37ºC (shaded histogram) or untreated (open histogram, left panel). The PBMC were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, and then stained with Alexa Fluor® 488 Mouse anti-4EBP1 (pT69, Cat. No. 560290). For data analysis, monocytes were selected by their scatter profile. The data demonstrates that the level of phosphorylation of 4EBP1 decreases when protein kinase activity is inhibited by the treatment. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
The specificity of mAb M34-273 was confirmed by western blot analysis (right panel) using unconjugated polyclonal anti-4EBP1 (Cell Signaling Technology, Cat. No. 9542, left blot) and unconjugated monoclonal Mouse anti-4EBP1 (pT69) (right blot) antibodies on lysates from control (lanes 1) and LY294002-treated (lanes 2) PBMC. 4EBP1 is identified as a band of 15-20 kDa in the left blot, regardless of LY294002 treatment. The right blot demonstrates the reduction of 4EBP1 (pT69) with LY294002 treatment (lane 2). Purified Mouse anti-Actin monoclonal antibody (Cat. No. 612656/612657) was the gel-loading control.