BD PhosflowTM Alexa Fluor 488 Mouse anti-4EBP1 (pT69)

Description

Analysis of 4EBP1 pT69 in human peripheral blood monocytes. Human peripheral blood mononuclear cells PBMC were either treated with 100 mu M 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 37C, 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 . Analysis of 4EBP1 pT69 in human peripheral blood monocytes. Human peripheral blood mononuclear cells PBMC were either treated with 100 mu M 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 37C, 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 . Show More Analysis of 4EBP1 pT69 in human peripheral blood monocytes. Human peripheral blood mononuclear cells PBMC were either treated with 100 mu M 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 37C, 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 .