Journal:
Article Title: The Hsp90-specific inhibitor geldanamycin selectively disrupts kinase-mediated signaling events of T-lymphocyte activation
doi: 10.1043/1355-8145(2000)005<0052:THSIGS>2.0.CO;2
Figure Lengend Snippet: Effect of geldanamycin (GA) on protein kinases involved in T-lymphocyte signaling. J32 cells (5 × 106 cells/mL) were incubated in the presence or absence of 1.78 μM GA at 37°C for 12 hours. Cells were serum starved during the last 4 hours of incubation and then stimulated with the T-cell receptor (TCR) ε-specific mAb OKT3 (OKT) at a final concentration of 5 μg/mL for 0 to 20 minutes as indicated. Cells were then washed and lysed in the presence of 1% Brij 98, and postnuclear supernatants of detergent extracts were prepared. In Western blot analysis (panels A, B, and D), 30 μg of postnuclear supernatant of detergent extracts were separated by 7.5% (panels A and B) or 10% (panel D) sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted with the respective antibodies as described in the text. In the MAPR kinase (MEK) kinase assay, 0.5 μg inactive ERK-2 was added to 5 μg of postnuclear supernatants of cell lysates and incubated in the presence of 50 μM [γ-32P]ATP for 20 minutes at 30°C as described in the text. (A) Effect of GA on Lck hyperphosphorylation. (B) Effect of GA on Raf-1 hyperphosphorylation. (C) Effect of GA on MEK activity as measured by the phosphorylation of inactive ERK-2. (D) Effect of GA on the endogenous ERK-2 hyperphosphorylation. Western blots and the autoradiogram are representatives of 5 independent experiments
Article Snippet: Recombinant, inactive ERK-2 was from New England Biolabs (Schwalbach/Taunus, Germany).
Techniques: Incubation, Concentration Assay, Western Blot, Polyacrylamide Gel Electrophoresis, Kinase Assay, Activity Assay
![Effect of geldanamycin (GA) on protein kinases involved in T-lymphocyte signaling. J32 cells (5 × 106 cells/mL) were incubated in the presence or absence of 1.78 μM GA at 37°C for 12 hours. Cells were serum starved during the last 4 hours of incubation and then stimulated with the T-cell receptor (TCR) ε-specific mAb OKT3 (OKT) at a final concentration of 5 μg/mL for 0 to 20 minutes as indicated. Cells were then washed and lysed in the presence of 1% Brij 98, and postnuclear supernatants of detergent extracts were prepared. In Western blot analysis (panels A, B, and D), 30 μg of postnuclear supernatant of detergent extracts were separated by 7.5% (panels A and B) or 10% (panel D) sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted with the respective antibodies as described in the text. In the MAPR kinase (MEK) kinase assay, 0.5 μg inactive <t>ERK-2</t> was added to 5 μg of postnuclear supernatants of cell lysates and incubated in the presence of 50 μM [γ-32P]ATP for 20 minutes at 30°C as described in the text. (A) Effect of GA on Lck hyperphosphorylation. (B) Effect of GA on Raf-1 hyperphosphorylation. (C) Effect of GA on MEK activity as measured by the phosphorylation of inactive ERK-2. (D) Effect of GA on the endogenous ERK-2 hyperphosphorylation. Western blots and the autoradiogram are representatives of 5 independent experiments](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2910/pmc00312910/pmc00312910__i1355-8145-005-01-0052-f05.jpg)