Fig 2: Inhibition of pY peptide/SH2 interaction by monobodies. (a) The graph shows relative pYEEI peptide binding (in %) to SH2 domain in the presence of a monobody. All eight SH2 domains have been measured without and in presence of the monobody selected for the respective on-target. The pYEEI peptide in isolation and the SH2/pYEEI complex were set to 0% and 100% binding, respectively. Accordingly, the reduction in binding observed in conjunction with a monobody is expressed as a percentage. Each data point corresponds to the average of at least two repeats +/- SD. (b) Relative peptide binding (in %) of three different peptides (pYEEI, pYQPQ, pYIIP) to the Lck SH2 domain in the presence of each of the two Lck monobodies [Mb(Lck_1) or Mb(Lck_3)]. The full sequence of the peptides can be found in the Materials and Methods section. (c) The monobodies Mb(Lck_3) or HA4-Y87A or buffer alone was added to biotinylated Lck or Src SH2 domains immobilized on Streptavidin-coated beads prior to incubation with lysate from stimulated Jurkat T cells. Immunoblot analysis after pull-down using an anti-pY antibody for a 40% fraction of beads (upper blot), using an anti-Zap70 antibody and an anti-phospho-Zap70 (pY319) antibody for a 40% fraction of beads (second and third blot from top), and using an anti-His-tag antibody to detect the recombinant SH2 domains and monobodies for a 10% fraction of beads (lower blot).

Fig 3: Enhanced proximal TCR signaling upon the expression of Zap70C564A. Zap70-deficient P116 T cells stably expressing Zap70wt, Zap70C564A, or no Zap70 (Zap70neg) were stimulated with the anti-CD3 (UCHT1) antibody for the indicated time periods. Subsequently, cells were lysed, and the phosphorylation of Zap70 on the regulatory tyrosines Y319 (A) and Y493 (B), and the phosphorylation of the ?-chain (Y142) (C), were investigated using phospho-specific antibodies. Equal protein loading was assessed using antibodies directed against the total Zap70 or CD3e. One representative experiment is shown (n = 6). Bands in (A–C) were quantified by normalizing the signal of phosphorylated Zap70 or ? to total Zap70 or CD3e, respectively. Graphs in (A–C) show the phosphorylation levels of the indicated molecules as arbitrary units ± SEM of six independent experiments. (D) HEK293T cells stably expressing the TCR or the TCR plus Lck were transiently transfected with plasmids encoding Zap70wt, Zap70C564A, or no Zap70 (Zap70neg). Subsequently, cell lysates were prepared and assayed for the phosphorylation of the ?-chain by immunoblotting. Immunoblotting using anti-CD3e was used to show equal loading. Bands in (D) were quantified by normalizing the signal of phosphorylated ? to the CD3e signal. The graph shows the phosphorylation levels of the ?-chain as arbitrary units ± SEM of four independent experiments. Significant p values were calculated using Welch’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001).

Fig 4: SYK transduces TLR4 signals leading to activation of ERK and AKT but not NF-?B. (A–C) Immunoblots of lysates from control and mutant B cells stimulated with LPS (A), CD40L (B), or LAS (C) for the indicated times. Blots were probed with antibodies to phospho-Ser473 AKT (pAKT), phospho-Thr202/Tyr204 ERK1/ERK2 (pERK), SYK, I?Ba, and ERK2. Graphs show mean ± SEM levels of pAKT, pERK, and I?Ba normalized to ERK2 and then to the levels in unstimulated control B cells (time 0), quantitated from multiple immunoblots. Minimum number of repeats per time point: A, 3 (pAKT), 9 (pERK), and 3 (I?Ba); B, 3 (pAKT), 7 (pERK), and 6 (I?Ba); C, 4 (pERK) and 4 (I?Ba). Numbers on righthand edge of all blots indicate molecular mass makers (kilodaltons). Statistical analysis was performed using two-way ANOVA (p-values for effect of genotype): *, P < 0.05; ****, P < 0.0001.

Fig 5: Constitutive active Lck enhances the phosphorylation and ZAP-70 association of RhoH in Jurkat T cells.Jurkat T cells transduced with a retroviral vector expressing HA-RhoH were transfected with combinations of the plasmids expressing EGFP, CA-Lck, kinase-dead Lck (KD-Lck), constitutive active ZAP-70 (AA-ZAP-70) or kinase-dead ZAP-70 (KA-ZAP-70). Total lysates were submitted to immunoprecipitation (IP) of anti-ZAP-70, anti-Lck or anti-HA Ab and immunoprecipitates and total lysates were analyzed by immunoblotting.