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Figure 3.
Biochemical and functional analysis of point mutants of SifA:
interaction with SKIP and RhoA, translocation, and formation of
Sifs. A, pulldown analysis of the interaction between SKIP(PH)
and SifA variants. GST::SKIP(PH) or GST were immobilized on
beads and incubated with extracts of HeLa cells expressing SifA,
SifA-(36–140), or SifA variants (AXXXE, WXXXA, AXXXA, and
L130D) fused to the N terminus of GFP or GFP alone. Bound
proteins were analyzed by Western blotting with an anti-GFP
antibody. B and C, translocation analysis. HeLa cells were
infected for 16 h with ΔsifA strains expressing GFP and
2HA-tagged version of wild-type or point-mutation variants of
SifA. Cells were either fixed, immunolabeled for HA, and imaged
by confocal microscopy for GFP (green) and HA (red) (scale bar,
10 μm) (B) or subjected to Triton X-100 extraction and
differential centrifugation and analyzed by Western blotting for
HA-tagged proteins in bacterial (BCT) and HeLa cell (HC)
fractions (C). D, both SifA and SifA-(L130D) pull down GDP-bound
RhoA. GST::SifA, GST::SifA-(L130D), or GST were immobilized on
beads and incubated with extracts of HeLa cells expressing
HA-tagged wild-type, GTP-bound (L63), or GDP-bound (N19) forms
of RhoA. Pulled down proteins were analyzed by Western blotting
with an anti-HA antibody. E, SifA-(L130D) does not support the
formation of Sifs. HeLa cells were infected for 16 h,
immunostained, and scored for the formation of HA-labeled Sifs.
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