Figure 1 Sample Clauses

Figure 1. Ras signaling via a Ral-dependent pathway is sufficient and required for ROS generation in T cells. A, Ras is sufficient and required for ROS generation in T lymphocytes. A total of 6 x 106 Jurkat cells were transfected by electroporation (250 V, 950 μF) with pCMV-CD20, to differentiate between transfected and untransfected cells, and empty pMT2HA expression plasmid (control) or cDNA encoding Ras signaling constructs indicated in each panel. At 48 h posttransfection, cells were stained with CyChrome-conjugated anti-CD20 mAbs. Cells were resuspended at 5 x 106 cells/ml in phenol red-free DMEM medium and loaded with 28 μM DCF for 20 min at 37°C before stimulation and ROS detection. Cells were stimulated with anti-CD3 ( ) or PMA/I (•) or left unstimulated ( ) and analyzed on a FACScan for the mean fluorescence intensity (MFI) of oxidated 6-carboxy-DCF in the FL1-channel, as a measurement for the presence of intracellular ROS, of CD20-expressing cells at different time points after stimulation. The ROS generation is obtained by reducing the measured MFI with the measured MFI at corresponding time points in the unstimulated, control-transfected Jurkat cells. The data shown are the mean values of three or more independent experiments. Expression of all constructs was confirmed by immunoblotting of cell lysates with anti-HA (for HA- RasV12 and HA-RasN17) or anti-Ras Abs (for RasV12, RasV12/G37, RasV12/E38, and RasV12/C40). B, Ral participates in TCR signaling. A total of 5 x 106 PB T lymphocytes from two healthy donors were stimulated for 5 min with either medium or anti-CD3 and anti–CD28 Abs ( CD3/CD28). GTP-bound Ral was precipitated, resolved by SDS-PAGE and visualized by immunoblotting with anti-Ral Abs and ECL (top). Total Ral expression was assessed by immunoblotting of whole cell lysates (bottom). C, Ral is sufficient and required for ROS generation downstream of Ras. Jurkat T cells were transfected with cDNA encoding Rlf, Ral, and Ras signaling mutants and assessed for ROS production as in A. Expression of Rlf-CAAX, RalV23, and RalN28 was assessed by immunoblotting of whole cell lysates with anti-HA Abs. Constructs used in A and B were assessed in the same experiments, but are split into two sections for clarity of presentation. Although our data clearly demonstrated that activation of endogenous Ral was required and sufficient for Ras-induced ROS generation, RasV12/G37 can also interact with PLCε (37), and overexpression of PLCε mimics RasV12/G37-induced...

Figure 1. (a) Superstrate-based 2T device stack. (b) Cross-sectional SEM image of a