BD PhosflowTM PE Mouse anti-4EBP1 (pT36/pT45)

Description

LEFT PANEL: Analysis of 4EBP1 pT36/pT45 in human peripheral blood monocytes. Human peripheral blood mononuclear cells PBMC were either treated with 100 muM LY294002 Sigma, Cat. No. L-9908 for 1 hour at 37oC shaded histogram or untreated open histogram . 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 PE Mouse anti-4EBP1 pT36/pT45 . 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. RIGHT PANEL: The specificity of mAb M31-16 was confirmed by western blot analysis using unconjugated polyclonal anti-4EBP1 Cell Signaling Technology, Cat. No. 9542, left blot and unconjugated monoclonal Mouse anti-4EBP1 pT36/pT45 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 pT36/pT45 with LY294002 treatment lane 2 . Purified Mouse anti-Actin monoclonal antibody Cat. No. 612656 or 612657 was the gel-loading control . LEFT PANEL: Analysis of 4EBP1 pT36/pT45 in human peripheral blood monocytes. Human peripheral blood mononuclear cells PBMC were either treated with 100 muM LY294002 Sigma, Cat. No. L-9908 for 1 hour at 37oC shaded histogram or untreated open histogram . 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 PE Mouse anti-4EBP1 pT36/pT45 . 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. RIGHT PANEL: The specificity of mAb M31-16 was confirmed by western blot analysis using unconjugated polyclonal anti-4EBP1 Cell Signaling Technology, Cat. No. 9542, left blot and unconjugated monoclonal Mouse anti-4EBP1 pT36/pT45 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 pT36/pT45 with LY294002 treatment lane 2 . Purified Mouse anti-Actin monoclonal antibody Cat. No. 612656 or 612657 was the gel-loading control . Show More LEFT PANEL: Analysis of 4EBP1 pT36/pT45 in human peripheral blood monocytes. Human peripheral blood mononuclear cells PBMC were either treated with 100 muM LY294002 Sigma, Cat. No. L-9908 for 1 hour at 37oC shaded histogram or untreated open histogram . 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 PE Mouse anti-4EBP1 pT36/pT45 . 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. RIGHT PANEL: The specificity of mAb M31-16 was confirmed by western blot analysis using unconjugated polyclonal anti-4EBP1 Cell Signaling Technology, Cat. No. 9542, left blot and unconjugated monoclonal Mouse anti-4EBP1 pT36/pT45 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 pT36/pT45 with LY294002 treatment lane 2 . Purified Mouse anti-Actin monoclonal antibody Cat. No. 612656 or 612657 was the gel-loading control .