SUPPLEMENTARY DATA LEGENDS
Supplementary Figure 1. Purity of the CD4+ T cells isolated from human cord
blood. This figure depicts the results of surface staining of the purified CD4+ T cells for
the CD3, CD4, CD8 and the CD45RA markers. Analysis was done by flow cytometry
and the histograms in black depict the corresponding profile obtained for unstained cells.
Figure shows best representative from at least three independent experiments.
Supplementary Figure 2. The effect of ERK-silencing on levels of intracellular IL-4
protein and transcription of IL-13, IL-10R, GATA3.
Panel A depict the effects of treatment of cells with ERK1/2 or GFP-specific siRNA on
levels of IL-4 protein expression. Here the IL-4 protein was detected by intracellular
staining (Methods) in cells obtained at day five of the activation (TCR)/polarization
(TCR+IL-4) culture. Panels B and C shows the corresponding effect of siRNA-mediated
silencing on the kinetics of IL-13 and IL-10R mRNA induction, respectively in cultured
cord blood-derived CD4+ T cells stimulated with TCR in the presence (TCR+IL-4) or
absence (TCR) of IL-4. The time points at which transcript levels were determined by
RT-PCR are indicated. In these samples, primers specific for -actin were also included
during amplification to provide an internal normalization control. The effects of ERK1/2versus GFP-silencing on GATA3 mRNA levels are shown in panel D. GATA3mRNA
expression was detected by RT-PCR of extracts obtained at 72h post stimulation (TCR+
IL-4). -actin was used as loading control. The figures show best representative from at
least two independent experiments. The effect of GFP (Black histogram) or ERK1/2
(Green histogram) siRNA-mediated silencing on cells expressing the IL-4R is shown in
panel E. Here the IL-4R was detected by FACS surface staining (Methods) in cells
obtained at day 3 and day 6 of the activation (TCR)/polarization (TCR+IL-4) culture.
This figure shows best representative from at least three independent experiments.
Supplementary Figure 3. Target specificity of U0126, ERK1 and ERK2 siRNA.
Panel A shows western blot profile of pERK1/2 in cells treated with/without U0126 (for
1h). PLC2 was used as loading control.
Panel B shows the effect and specificity of ERK1 and ERK2 siRNAs (western blot
profiles). M represents mock transfected sample while E1, E2 and E1/E2 represent ERK1
or ERK2 siRNA or both silenced cells, respectively. GAPDH was used as loading
control.
Supplementary Figure 4.
Panel A shows the western blot profiles obtained for ERK1/2 and GAPDH in the
cytoplasmic fraction of lysates of purified naive CD4+ T cells. Here, the results for
samples obtained from thirty eight different individuals (numbered 1-38) are shown.
Detection and quantification of band intensities was done using Odyssey software version
3.0. Panel B shows the western blots profiles obtained for both the phosphorylated form
of ERK1/2 (p-ERK1/2) and the ERK1/2 protein (ERK) in lysates of purified CD4+ T
cells that had been stimulated under Th2 polarizing conditions for 3h. Results obtained in
cells from twenty different individuals (numbered 1-20; U=unstimulated) are shown (see
Text for details). GAPDH was used as loading control.
Supplementary Table 1. Nucleotide sequence of siRNA and primers.
Table in panel A identifies the target sequences within ERK-1 and ERK-2 mRNA against
which the siRNAs from the indicated sources were designated. Panel B provides the
nucleotide sequences used for the RT-PCR of the indicated target mRNAs. Sequence of
Chip Primers are shown in panel C.