Ectopically expressed MyoD interacts with endogenous HP1 proteins.
A, silver staining the double affinity-purified MyoD complex isolated
from chromatin fractions in a HeLa cell line stably expressing FLAG-HA tagged
MyoD. MW, protein molecular weight marker. The molecular masses of
the markers are indicated. B, Western blot analysis of double
purified FLAG-HA-MyoD (MyoD) or eluate from HeLa control cells
(Mock). C, top panel, Western blotting with anti-MyoD on
streptavidin-bead precipitates from HEK 293 cell extracts transfected with a
MyoD expression vector (lanes 1–4) or with an empty vector
(lanes 5–7), along with expression vectors for the biotinylated
forms of HP1α (bHP1α)(lanes 3 and 7), HP1β
(lanes 2 and 6), HP1γ (lanes 1 and
5), or the empty vector (lane 4). Middle and
bottom panels, quantity control of MyoD detected with an anti-MyoD
antibody (middle panel) and biotinylated HP1 detected using
streptavidin-horseradish peroxidase conjugate (bottom panel) in the
inputs. D, top panel, Western blotting of anti-FLAG-HP1 of
streptavidin bead precipitates from HEK 293 cell extracts transfected with the
expression vector for FLAG-HP1α (lanes 1 and 4),
FLAG-HP1β (lanes 2 and 5), HP1γ (lanes 3 and
6), or with the empty vector (lane 7), along with expression
vectors for the biotinylated form of MyoD (lanes 4–7) or the
empty vector (lanes 1–3). Middle and bottom
panels, quantity control of biotinylated MyoD (bMyoD) detected
using streptavidin-horseradish peroxidase conjugate (middle panel)
and that of FLAG-HP1 protein in the inputs as detected with an anti-FLAG
antibody (bottom panel). *, endogenous biotinylated proteins.
IB, immunoblot.