Intracellular signaling events downstream of EphB6. Bar graphs show the density ratios of phosphorylated molecules versus total protein (as loading controls) of the molecule. (A) EphB6 was required for optimal LAT phosphorylation. EphB6^+/+ or EphB6^–/– thymocytes were cross-linked with anti-CD3 and anti-CD4 mAbs for 0–5 minutes, as indicated. The cell lysates were immunoblotted (Blot) with Ab against phosphorylated LAT (p-LAT) first (upper panel); the membrane was stripped and reblotted with Ab against LAT (lower panel). (B) EphB6 was required for optimal ZAP-70 phosphorylation. EphB6^+/+ or EphB6^–/– thymocytes were cross-linked with anti-CD3 and anti-CD4 mAbs for 0 minutes (resting) or 1 minute (activated). The cell lysates were immunoblotted (Blot) with Ab against phosphorylated ZAP-70 (p-ZAP-70) first (upper panel); the membrane was stripped and reblotted with Ab against ZAP-70 (lower panel). (C) EphB6 was not necessary for ZAP-70 membrane translocation. EphB6^+/+ or EphB6^–/– thymocytes were cross-linked with anti-CD3 and anti-CD4 for 0 minutes (resting) or 20 minutes (activated). The whole-cell lysates, cytosolic protein, and membrane protein, as indicated, were immunoblotted with Ab against ZAP-70. (D) EphB6 augmented binding of SLP-76 with PLCγ1 after TCR activation. EphB6^+/+ or EphB6^–/– thymocytes were cross-linked with anti-CD3 and anti-CD4 mAbs for 2 minutes. The lysates were immunoprecipitated (IP) with anti-PLCγ1 and blotted with anti–SLP-76 (upper panel). The same membrane was stripped and blotted again with anti-PLCγ1 (lower panel). (E) EphB6 was required for optimal Erk1/2 activation. EphB6^+/+ or EphB6^–/– thymocytes were cross-linked with anti-CD3 and anti-CD4 mAbs for 0–5 minutes, as indicated. The cell lysates were immunoblotted with Ab against phosphorylated Erk1/2 first (p-Erk1/2, upper panel); the same membrane was stripped and reblotted with Ab against Erk1/2 (lower panel).