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. 2005 May;25(10):4117–4128. doi: 10.1128/MCB.25.10.4117-4128.2005

FIG. 1.

FIG. 1.

CD28 and CTLA-4 have opposing actions on ERKs and Rap1. (A) In vitro-primed AD10 T cells were left untreated (Un) or treated for 5 min with immobilized anti-CD3 (CD3), anti-CD28 (CD28), and/or anti-CTLA-4 (CTLA-4) as indicated. Cells extracts were analyzed by immunoblotting with antibodies specific for phosphorylated ERK1/2 (pERK1/2). The levels of total ERK2 are indicated as a loading control. (B) In vitro-primed AD10 T cells were left untreated (Un) or treated for 5 min with immobilized anti-CD3 (CD3), anti-CD28 (CD28), or anti-CTLA-4 (CTLA-4). Activated Rap1 (Rap1-GTP) and Ras (Ras-GTP) were precipitated using GST-RalGDS and GST-Raf-1GDS, respectively, and analyzed by immunoblotting with Rap1- or Ras-specific antibodies. Total levels of Rap1 are indicated as loading controls for both assays. (C) In vitro-primed AD10 T cells were left untreated (Un) or treated for the indicated times with immobilized anti-CTLA-4 (CTLA-4). Activated Rap1 (Rap1-GTP) was pulled down using GST-RalGDS and analyzed by immunoblotting with Rap1-specific antibodies (Ab). Total levels of Rap1 are indicated as a loading control.