M-CSF and GM-CSF Regulation of STAT5 Activation and DNA Binding in Myeloid Cell Differentiation is Disrupted in Nonobese Diabetic Mice
Figure 3
STAT5 binding to chromatin increases after GM-CSF stimulation in NOD but not C57BL/6 bone marrow cells. Five million bone marrow cells were cultured
with or without 100M Na vanadate for 30 minutes at 37/5.
Half of the cultures vanadate were then supplemented with 1000?U/mL GM-CSF, and all were
incubated for an additional 90 minutes at 37/5. The cells were then fixed in situ, extracted, and sonicated.
The sample was split into five cell aliquots for use in
anti-STAT5Ptyr/anti-Histone H3-mediated double ChIP protein isolations for Western
blot analysis of STAT5 associated with histone/chromatin complexes. Protein
isolated from the precipitated chromatin complexes and analyzed by Western blot
probed with anti-STAT5Ptyr antibody. Densitometric/Rf-value anti-STAT5Ptyr Western blot-detected bands separated on 4-20% SDS-PAGE were used to give the approximate
size and location of the STAT5 protein complexes and monomers (approximate
molecular weights indicated on the left of the figure). Higher molecular weight
bands that bound the anti-STAT5Ptyr antibody had Rf values suggesting that they
may represent a mix of formaldehyde-cross-linked dimer complexes containing mixed isoforms of STAT5 (STAT5A (A
96?K), STAT5B (B 92?K), and truncated (T 80?K, 77?K)), both in homodimers (A-A
192?K, B-B 184?K, T-T) and heterodimers (A-B, A-T, B-T, T-T) complexes. Only
monomeric STAT5A size bands (96?K) are detected in the GM-CSF-induced NOD cell
cultures at this early time point. The positions of the precipitating
antibodies heavy chain (50?K) and light chain (25?K), which acts as an internal
protein loading standards, were determined from the extract minus sham control
(sh) and size standards
(indicated but not shown). Treatment/lane key: 0 = untreated, V =
vanadate alone, G = GM-CSF
alone, VG = vanadate and GM-CSF-supplemented
2-hour cultures. Data represents 3 runs
of the double ChIP analysis.
