2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:50 PM Page 7
To develop more mechanistic insight into the aberrant ability of IFN-α to A similar pattern of prolonged Stat1 activation in Stat2-/- macrophages was
induce MCH II expression in Stat2-/- macrophages, extracts were prepared observed by EMSA. Specifically in WT macrophages, IFN-α induced a rapid and
from IFN stimulated wild type and Stat2-/- macrophages, and then evaluated transient activation of ISGF-3 (Stat1 + Stat2 + IRF-9), which could be super-
for STAT activity by immunoblotting and electrophoretic mobility shift assay shifted with a Stat2-specific antibody (3, 9, 10). IFN-αalso transient stimulated
(EMSA). As previously reported in WT macrophages, IFN-α directed both the GAS binding activity (i.e., a Stat1:Stat1 homodimer) in WT macrophages (Fig.
rapid (i.e., within 0.5 h) and transient (i.e., peaking at 0.5 h) activation (i.e., 2B, upper panels). In contrast, in Stat2-/- macrophages, IFN-α stimulated
tyrosine phosphorylation) of Stat1 and Stat2 (Fig. 2A). In contrast, IFN-γ prolonged GAS binding activity, analogous to the pattern observed in IFN-γ
directed a strong and prolonged pattern of Stat1 activation, with no effect on treated cells (Fig. 2B, lower panels). Again, this activity could be super shifted
Stat2 (Fig. 2A). Intriguingly, the pattern of Stat1 activation was markedly with a Stat1-specific antibody. These results suggest that the transient pattern
prolonged in IFN-α stimulated Stat2-/- macrophages (Fig. 2A). of IFN-αstimulated Stat1 activation observed in WT macrophages is dependent
on the presence of Stat2.
Figure 2: IFN dependent signaling in wild type and Stat2-/- BM
Figure 2
macrophages.
(A)Whole cell extracts (WCE) were prepared from WT and Stat2
-/-
BMMs, either before or after stimulation with IFN-α
A/D
(1000 U/ml)
or IFN-γ (66 U/ml), as indicated. Samples were fractionated by SDS-
PAGE and immunoblotted with antibodies specific for phospho-Stat1,
phospho-Stat2 and actin, as indicated.
(B) WCEs, prepared as in panel A, were evaluated by EMSA with ISRE
and GAS probes. In the left most panel, extracts from IFN-αA/D
treated WT BMMs were super-shifted by the addition of either
preimmune serum (PI), Stat2 (upper) or Stat1 (lower)-specific
antibody (Ab), 30 min prior to incubation with probe. The mobility of
ISGF3 and Stat1 homodimers (St1:1) are indicated.
(C)Q-PCR, with Socs-1 and Socs-3 specific primers, was carried out
on total RNA (5 µg) from WT and Stat2
-/-
BMMs either before or
after stimulation with IFN-αA/D (1000 U/ml) or IFN-γ (66 U/ml), as
indicated. Socs-1 and Socs-2 expression were normalized to that
of β-actin.
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