BD PhosflowTM PE Mouse anti-Human IKKgamma (pS376)

Description

Analyses of IKKgamma pS376 expression. Panel 1: Multicolor flow cytometric analysis of IKKgamma pS376 expression by human T lymphocytes. Human whole blood collected with heparin was either left unstimulated dashed line histogram or stimulated solid line histogram with 400 nM PMA and 250 ng/ml Ionomycin Sigma, Cat No. I-0634 at 37C for 10 minutes. Cells were fixed in 1X BD Phosflow Lyse/Fix Buffer Cat. No. 558049 for 5X stock at 37C for 10 min. The cells were permeabilized in BD Phosflow Perm Buffer III Cat. No. 558050 on ice for at least 30 min or overnight at -20C , washed and then stained with BD Phosflow PE Mouse anti-Human IKKgamma pS376 Cat. No. 562590 and APC Mouse Anti-Human CD3 Cat. No. 561810/555335/561811 antibodies. The fluorescence histograms were derived from CD3 positive-gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry was performed using a BD FACSCanto II Flow Cytometer System. Panel 2: Western blot analysis of IKKgamma pS376 in human epithelioid carcinoma. HeLa S3 cells ATCC CCL 2.2 were cultured overnight in medium containing 0.1 % FBS and then either not treated C or stimulated T with recombinant human Tumor Necrosis Factor Cat. No. 554618; 25 ng/ml and Calyculin A Calbiochem Cat. No. 208851; 50 nM at 37C for 10 minutes. Lysates 15 mug total cell protein/lane were blotted using Purified Mouse Anti-Human IKKgamma pS376 antibody Cat. No. 562589; at 1.0, 0.5 and 0.25 mug/ml for Lanes 1, 2, 3, respectively , HRP Goat Anti-Mouse Ig Cat. No. 554002 and a chemiluminescent detection system. Phosphorylated IKKgamma proteins were identified as 50-55 kDa bands by blotting. Panel 3: Western blot analysis of IKKgamma pS376 expressed by human peripheral blood mononuclear cells PBMC . Human PBMC were stimulated with 50 nM Phorbol 12-Myristate 13-Acetate PMA, Sigma, Cat. No. P-8139 at 37C for 10 minutes, and lysates were analyzed by Western blotting as described above. Analyses of IKKgamma pS376 expression. Panel 1: Multicolor flow cytometric analysis of IKKgamma pS376 expression by human T lymphocytes. Human whole blood collected with heparin was either left unstimulated dashed line histogram or stimulated solid line histogram with 400 nM PMA and 250 ng/ml Ionomycin Sigma, Cat No. I-0634 at 37C for 10 minutes. Cells were fixed in 1X BD Phosflow Lyse/Fix Buffer Cat. No. 558049 for 5X stock at 37C for 10 min. The cells were permeabilized in BD Phosflow Perm Buffer III Cat. No. 558050 on ice for at least 30 min or overnight at -20C , washed and then stained with BD Phosflow PE Mouse anti-Human IKKgamma pS376 Cat. No. 562590 and APC Mouse Anti-Human CD3 Cat. No. 561810/555335/561811 antibodies. The fluorescence histograms were derived from CD3 positive-gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry was performed using a BD FACSCanto II Flow Cytometer System. Panel 2: Western blot analysis of IKKgamma pS376 in human epithelioid carcinoma. HeLa S3 cells ATCC CCL 2.2 were cultured overnight in medium containing 0.1 % FBS and then either not treated C or stimulated T with recombinant human Tumor Necrosis Factor Cat. No. 554618; 25 ng/ml and Calyculin A Calbiochem Cat. No. 208851; 50 nM at 37C for 10 minutes. Lysates 15 mug total cell protein/lane were blotted using Purified Mouse Anti-Human IKKgamma pS376 antibody Cat. No. 562589; at 1.0, 0.5 and 0.25 mug/ml for Lanes 1, 2, 3, respectively , HRP Goat Anti-Mouse Ig Cat. No. 554002 and a chemiluminescent detection system. Phosphorylated IKKgamma proteins were identified as 50-55 kDa bands by blotting. Panel 3: Western blot analysis of IKKgamma pS376 expressed by human peripheral blood mononuclear cells PBMC . Human PBMC were stimulated with 50 nM Phorbol 12-Myristate 13-Acetate PMA, Sigma, Cat. No. P-8139 at 37C for 10 minutes, and lysates were analyzed by Western blotting as described above. Show More Analyses of IKKgamma pS376 expression. Panel 1: Multicolor flow cytometric analysis of IKKgamma pS376 expression by human T lymphocytes. Human whole blood collected with heparin was either left unstimulated dashed line histogram or stimulated solid line histogram with 400 nM PMA and 250 ng/ml Ionomycin Sigma, Cat No. I-0634 at 37C for 10 minutes. Cells were fixed in 1X BD Phosflow Lyse/Fix Buffer Cat. No. 558049 for 5X stock at 37C for 10 min. The cells were permeabilized in BD Phosflow Perm Buffer III Cat. No. 558050 on ice for at least 30 min or overnight at -20C , washed and then stained with BD Phosflow PE Mouse anti-Human IKKgamma pS376 Cat. No. 562590 and APC Mouse Anti-Human CD3 Cat. No. 561810/555335/561811 antibodies. The fluorescence histograms were derived from CD3 positive-gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry was performed using a BD FACSCanto II Flow Cytometer System. Panel 2: Western blot analysis of IKKgamma pS376 in human epithelioid carcinoma. HeLa S3 cells ATCC CCL 2.2 were cultured overnight in medium containing 0.1 % FBS and then either not treated C or stimulated T with recombinant human Tumor Necrosis Factor Cat. No. 554618; 25 ng/ml and Calyculin A Calbiochem Cat. No. 208851; 50 nM at 37C for 10 minutes. Lysates 15 mug total cell protein/lane were blotted using Purified Mouse Anti-Human IKKgamma pS376 antibody Cat. No. 562589; at 1.0, 0.5 and 0.25 mug/ml for Lanes 1, 2, 3, respectively , HRP Goat Anti-Mouse Ig Cat. No. 554002 and a chemiluminescent detection system. Phosphorylated IKKgamma proteins were identified as 50-55 kDa bands by blotting. Panel 3: Western blot analysis of IKKgamma pS376 expressed by human peripheral blood mononuclear cells PBMC . Human PBMC were stimulated with 50 nM Phorbol 12-Myristate 13-Acetate PMA, Sigma, Cat. No. P-8139 at 37C for 10 minutes, and lysates were analyzed by Western blotting as described above.