Skip to main content
. 2015 Oct 2;1(9):e1500415. doi: 10.1126/sciadv.1500415

Fig. 2. Mono-7D6-Fab does not stimulate early TCR/CD3 signaling events but induces CD3Δc.

Fig. 2

(A) B6 cells were treated with Mono-7D6-Fab on ice versus 37°C with or without pervanadate (PV) for 5 min. Lysates were immunoprecipitated with control IgG or anti-CD3ε (APA1/1). CD3-associated phosphotyrosine was detected by Western blot (WB) analysis using the anti-PY (phospho-tyrosine) mAb 4G10 (short and long exposures shown). TL, total lysate content before immunoprecipitation (Ip). (B) Indo1/AM-loaded B6 cells were stimulated with the indicated reagents for 5 min at 37°C. Calcium flux was measured as a ratio of 405-nm/495-nm fluorescence gated on Thy1.2+ T cells. (C) Pmel-1 lysate was incubated with APA1/1 (pull-down blocking condition), hamster IgG (basal condition), 2C11 (full induction condition), or Mono-7D6-Fab (test condition). Lysates were subjected to CD3-PD assay, and open CD3 conformation was determined by WB analysis of the CD3ζ subunit. Numbers represent a fold increase in PRS-accessible (open) CD3. TL, total lysate content before pull-down.