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Fig. 5. HDGF properties and associations revealed through sequence similarities and
isolation of a nuclear multi-protein complex. (A) Rapid release of nuclear
HDGF but not histone 2Az protein following treatment of cultured HeLa cells
with low concentrations of NP-40 detergent. This behavior parallels that of
the related protein HMGB1 (Scaffidi et
al., 2002) and may explain why HDGF previously was found intact in
conditioned culture media. (B) Silver-stained gel of HDGF immunoprecipitates
from mock-transfected or epitope-tagged HDGF-overexpressing HeLa cells
resolved by SDS-PAGE. The marked specific bands were extracted and identified
by mass spectrometric analysis to contain the indicated proteins: two novel
factors (Fig. S1), hnRNPs K and I, and TLS/Fus, also known as mouse pigpen.
(C) Immunoblot analysis of HeLa cell (top) and E13 mouse intestine (bottom)
nuclear fractions resolved over a glycerol gradient and probed for the
presence of HDGF, hnRNPK and TLS/Fus proteins. Resolution of protein complexes
is similar in the two cell sources and indicates that a significant proportion
of nuclear HDGF may be complexed with hnRNPK (red) and TLS/Fus (green), which
in turn may associate independently with each other and with other factors in
bulkier protein complexes. (D) Immunofluorescence analysis of subcellular
localization of HDGF and TLS/Fus (similar results for hnRNPK are not shown)
reveals the abundance of each in tiny nuclear dots, although the resolution
cannot unambiguously define protein association in this context. (E) mRNA
(RT-PCR, left) and protein (immunoblot, right) levels of the putative
HDFG-associated factors TLS/Fus and hnRNPs K and I are downregulated in tandem
with HDGF during mouse intestine development. These results reinforce the
possibility of the four proteins functioning within a common cellular pathway
that regulates epithelial differentiation and cancer. N.T., not tested.
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