BD PhosflowTM Alexa Fluor 647 Mouse anti-S6 (pS244)

Description

Analysis of S6 pS244 in activated human peripheral blood mononuclear cells PBMC . PBMC were isolated by density gradient centrifugation Ficoll-Paque PLUS, Cat. No. 17-1440-02 and either left untreated open histogram or treated with PMA Sigma-Aldrich, Cat. No. P8139 at 50 nM/106 cells for 30 minutes shaded histogram . Cells were then fixed in BD Cytofix buffer Cat. No. 554655 at 37C for 10 minutes, then permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 on ice for at least 30 minutes, and then stained with Alexa Fluor 647 Mouse anti-S6 pS244, Cat. No. 560645 . For data analysis, lymphocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSCalibur flow cytometry system. Western blot analysis of S6 pS244 . The specificity of mAb N5-676 was confirmed by western blot analysis using unconjugated Mouse anti-S6 pS244 antibody on lysates from untreated lane 1 or PMA-treated lane 2 PBMC. S6 pS244 is identified as a band of 32 kDa, with increased intensity in the PMA-treated cells. Quantitative western blot analyses demonstrated that the unidentified high-molecular-weight bands are not induced by the PMA treatment data not shown and, thus, do not contribute to the signal increase that is observed by flow cytometry. Purified Mouse anti-Actin monoclonal antibody Cat. No. 612656/612657 was the gel-loading control. Analysis of S6 pS244 in activated human peripheral blood mononuclear cells PBMC . PBMC were isolated by density gradient centrifugation Ficoll-Paque PLUS, Cat. No. 17-1440-02 and either left untreated open histogram or treated with PMA Sigma-Aldrich, Cat. No. P8139 at 50 nM/106 cells for 30 minutes shaded histogram . Cells were then fixed in BD Cytofix buffer Cat. No. 554655 at 37C for 10 minutes, then permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 on ice for at least 30 minutes, and then stained with Alexa Fluor 647 Mouse anti-S6 pS244, Cat. No. 560645 . For data analysis, lymphocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSCalibur flow cytometry system. Show More Western blot analysis of S6 pS244 . The specificity of mAb N5-676 was confirmed by western blot analysis using unconjugated Mouse anti-S6 pS244 antibody on lysates from untreated lane 1 or PMA-treated lane 2 PBMC. S6 pS244 is identified as a band of 32 kDa, with increased intensity in the PMA-treated cells. Quantitative western blot analyses demonstrated that the unidentified high-molecular-weight bands are not induced by the PMA treatment data not shown and, thus, do not contribute to the signal increase that is observed by flow cytometry. Purified Mouse anti-Actin monoclonal antibody Cat. No. 612656/612657 was the gel-loading control. Show More Analysis of S6 pS244 in activated human peripheral blood mononuclear cells PBMC . PBMC were isolated by density gradient centrifugation Ficoll-Paque PLUS, Cat. No. 17-1440-02 and either left untreated open histogram or treated with PMA Sigma-Aldrich, Cat. No. P8139 at 50 nM/106 cells for 30 minutes shaded histogram . Cells were then fixed in BD Cytofix buffer Cat. No. 554655 at 37C for 10 minutes, then permeabilized with BD Phosflow Perm Buffer III Cat. No. 558050 on ice for at least 30 minutes, and then stained with Alexa Fluor 647 Mouse anti-S6 pS244, Cat. No. 560645 . For data analysis, lymphocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSCalibur flow cytometry system. Western blot analysis of S6 pS244 . The specificity of mAb N5-676 was confirmed by western blot analysis using unconjugated Mouse anti-S6 pS244 antibody on lysates from untreated lane 1 or PMA-treated lane 2 PBMC. S6 pS244 is identified as a band of 32 kDa, with increased intensity in the PMA-treated cells. Quantitative western blot analyses demonstrated that the unidentified high-molecular-weight bands are not induced by the PMA treatment data not shown and, thus, do not contribute to the signal increase that is observed by flow cytometry. Purified Mouse anti-Actin monoclonal antibody Cat. No. 612656/612657 was the gel-loading control.