Figure 4. Overexpression of... | Rockefeller University Press

Figure 4.

$Figure 4. Overexpression of p50-dynamitin-GFP impairs sustained signaling via the TCR. (A–E) Western analysis of signaling proteins in J77-GFP or dynamitin-disrupted cells upon stimulation with SEE for the times indicated. Whole lysates were processed for phosphorylated and total protein expression as follows: (A) ZAP70 pY315 and total ZAP70; (B) LAT pY132, pY171, pY191, pY226, and total LAT; (C) Vav1 pY174 and total Vav1; (D) PKCθ pT538 and total PKCθ; and (E) phosphorylated and total Erk1/2, followed by the corresponding HRP-conjugated secondary antibodies. One out of four representative experiments is shown. Arrows indicate target band sizes. (F–H) Conjugates formed and stained for proteins involved in IS formation and maturation (red in merged images, Alexa 568–labeled anti–mouse secondary antibody): LAT (F; arrowheads indicate the fraction of LAT associated with the nontranslocated MTOC in the dynamitin-disrupted clones and not delivered to the nascent IS), pY174Vav1 (G), and PKCθ (H). Arrows indicate the IS area. R, SEE-Raji APC (CMAC-labeled, blue). Bars, 10 μm.$

Overexpression of p50-dynamitin-GFP impairs sustained signaling via the TCR. (A–E) Western analysis of signaling proteins in J77-GFP or dynamitin-disrupted cells upon stimulation with SEE for the times indicated. Whole lysates were processed for phosphorylated and total protein expression as follows: (A) ZAP70 pY315 and total ZAP70; (B) LAT pY132, pY171, pY191, pY226, and total LAT; (C) Vav1 pY174 and total Vav1; (D) PKCθ pT538 and total PKCθ; and (E) phosphorylated and total Erk1/2, followed by the corresponding HRP-conjugated secondary antibodies. One out of four representative experiments is shown. Arrows indicate target band sizes. (F–H) Conjugates formed and stained for proteins involved in IS formation and maturation (red in merged images, Alexa 568–labeled anti–mouse secondary antibody): LAT (F; arrowheads indicate the fraction of LAT associated with the nontranslocated MTOC in the dynamitin-disrupted clones and not delivered to the nascent IS), pY174Vav1 (G), and PKCθ (H). Arrows indicate the IS area. R, SEE-Raji APC (CMAC-labeled, blue). Bars, 10 μm.