Fig 2: Effect of Lck and Fyn ectopic expression in Th2 cells on Y174 VAV1 phosphorylation. Naive CD4+ cells were transduced (A only) with control, Lck and Fyn encoding retroviruses and polarized (A,B) under Th2 or Th1 (control vector only) conditions. 5 days after initiation of cell cultures GFP+ (A) or total (B) cells were isolated, rested overnight without APCs, antibodies and cytokines and re-stimulated (B only) with anti-CD3 (10 µg/ml) and anti-CD28 (2 µg/ml) antibodies. Western blotting of total cell lysate (A) or of cytoplasmic/cell membrane fraction (B). (A) Cropped blots are shown. Complete blots are shown in Supplementary Fig. S9. Staining was performed using the same blots as in Fig. 2A. Correspondingly, the same β-Actin staining is shown there as loading control. Results of a representative experiment of two experiments are shown.

Fig 3: Reduced Lck and CD4 expression in mouse Th2 cells. Naive CD4+ T cells were polarized under Th1 and Th2 conditions for 5 days, rested overnight without APCs, antibodies and cytokines and re-stimulated with anti-CD3 (10 µg/ml) and anti-CD28 (2 µg/ml) antibodies. (A,D) Western blotting analysis of cytoplasmic/cell membrane fraction (A) or total cell lysate (D). Results of a representative experiment of four experiments are shown. (B) Densitometry analysis of Western blot images of resting Th1 and Th2 cells. Average and standard deviation of three (pY394Lck/total Lck ratio) and seven (total Lck/β-Actin and total Fyn/β-Actin ratios) experiments are shown. Mann Whitney U test was used to perform statistical comparisons (only for total Lck/β-Actin and total Fyn/β-Actin ratios). (C) Flow cytometry analysis of Th1 and Th2 cells. Results of a representative experiment (of four experiments) are shown. (E) Lck mRNA expression in Th1 and Th2 cells. Lck V1 and Lck V2–transcripts from the proximal promoter, Lck V3–transcript from the distal promoter. Average and standard deviation of eight independent experiments are shown. Student's t-test was used to perform statistical comparisons.

Fig 4: Ectopic expression of Lck and Fyn in Th2 cells increases S73 phosphorylation of c-Jun. Naive CD4+ cells were transduced and polarized as described in the legend to Fig. 2. 5 days after initiation of cell culture GFP+ cells were isolated, rested overnight without APCs, antibodies and cytokines and re-stimulated with anti-CD3 and anti-CD28 antibodies. (A) Western blot of nuclear fraction of cell re-stimulated with soluble anti-CD3 and anti-CD28 antibodies. Results of a representative experiment of three experiments are shown. (B) Densitometry analysis of Western blot images of pS73 and total c-Jun from (A). Signals obtained from pS73 c-Jun blots were divided to signals of total c-Jun blots and normalized to the mean signals ratio for each experiment. Average and standard deviation of three experiments are shown. Densitometry data used for generation of histogram are shown in Supplementary Table S2. (C) Flow cytometry analysis of GFP+ cells re-stimulated with immobilized anti-CD3 and soluble anti-CD28 antibodies. Inhibitors of JNK (SP600125, 50 µM) and ERK (U0126, 10 µM) cascades are added 1 hour before cells re-stimulation. Results of a representative experiment of three experiments are shown.