Figure 6. Vav1^ΔC‐mediated proliferation requires engagement of several downstream pathways.

• A
  Schematic representation of the experiments used in this figure. See details in main text and Materials and Methods.
• B
  Depiction of the Vav1 mutants used in these experiments. The E201A mutation is shown as a gray circle. The activity of each mutant is represented on the right. Green box, WT activity; red box, gain of function; blue box, loss of function.
• C
  Representative immunoblot showing the abundance of the ectopic Vav1 proteins, EGFP, and the endogenous tubulin α (loading control) in CD4⁺ T cells transduced with retrovirus particles used in these experiments (top).
• D
  Representative FACS plots showing EGFP epifluorescence in CD4⁺ T cells transduced with retroviruses encoding the indicated proteins (top). Numbers indicate the relative percentage (%) of the EGFP⁺ cell population in each case. Gray shaded histograms represent the fluorescence obtained from CD4⁺ T cells nontransduced with retrovirus particles. Similar results were obtained in CD4⁺ T cells transduced with virus encoding EGFP (n = 6), Vav1^WT (n = 6), Vav1^ΔC (n = 6), Vav1^ΔC+E201A (n = 6), and Vav1^ΔN (n = 3).
• E, F
  Representative FACS plots (E) and quantification (F) of the proliferation of EGFP⁺ CD4⁺ T cells expressing the indicated proteins using the Cell Trace Violet method. In (E), the gray shaded histograms represent the fluorescence obtained from nonstimulated CD4⁺ T cells before stimulation and retroviral transduction. In (F), each point represents the values obtained with a single experimental mouse. n = 6 per each experimental condition, except in the case of Vav1^ΔN (n = 3).
• G–J
  Flow cytometry determination of Tox (G), ICOS (H), CD25 (I), and ICN1 (J) expression in EGFP⁺ CD4⁺ T cells expressing the indicated Vav1 proteins. NSt, nonstimulated cells (CD4⁺ T cells before stimulation and retroviral transduction). In all panels, each point represents the values obtained for a single experimental condition. f.i., mean fluorescence intensity relative to the isotype‐matched control antibody. n = 6 per each experimental condition, except for the Vav1^ΔN and NSt conditions (n = 3).
• K–N
  Example of a flow cytometry analysis (K and M) and final quantification of p‐Akt (L) and p‐Erk1/2 (N) levels in EGFP⁺ CD4⁺ T cells expressing the indicated Vav1 proteins in primed (blue bars) and restimulated (red bars) cells. In K and M, the gray shaded histograms represent the fluorescence obtained with the isotype‐matched control antibody. In L and N, NSt, nonstimulated, St, stimulated. n = 3 per each experimental condition.

Data information: In panels (F, G, H, I, J, L, and N), values are shown as means ± SEM from three independent experiments. In panels L and N, P‐values are given relative to nonstimulated (blue asterisks) and stimulated (red asterisks) cells expressing Vav1^WT. We also included P‐values for the values exhibited by each Vav1 mutant protein relative to those obtained in nonstimulated condition (black asterisks). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 (Student’s t‐test).

Source data are available online for this figure.