SDF2L1 interacts with proHNP3 and the mature HNP3 sequence is sufficient
for this interaction. A, schematic representations of the
DBD-fusion constructs used in this figure. B, spot tests were
performed using three colonies of AH109 cells transformed with plasmids
DBD-proHNP3 and AD-SDF2L1, DBD-proHNP3, and AD-ΔN28SDF2L1,
and the controls: DBD-proHNP3 and AD-vector, and DBD-vector and
AD-ΔN28SDF2L1 as indicated in the figure. C, In
vitro interaction between proHNP3 and SDF2L1. Left panel,
SDS-tricine PAGE of GST (lane 1) and GST-proHNP3 (lane 2)
purified from yeast extracts with glutathione-Sepharose beads and then
Coomassie Blue-stained. Right panel, GST (lane 2) and
GST-proHNP3 columns (lane 3) were incubated with
[35S]HA-SDF2L1. Bound proteins were analyzed by autoradiography.
Lane 1, input [35S]HA-SDF2L1 (arrowhead).
D, spot tests of AH109 cells transformed with plasmids listed
(Table 1). E, in vitro
interaction between HNPs and SDF2L1; left panel, Coomassie
Blue-stained SDS-Tricine PAGE of GST (lane 1) and
GST-ΔN28SDF2L1 (lane 2) prepared as in C
above. Right panel, GST (lane 1) and
GST-ΔN28SDF2L1 (lane 2) columns incubated with
metabolically labeled HL60 extracts. Bound proteins were analyzed by
autoradiography. Control immunoprecipitations with labeled HL60 extracts and
protein A-Sepharose beads and either rabbit preimmune IgG (lane 3) or
α-HNP IgG (lane 4). The arrowhead indicates
35S-labeled HNP, and the asterisk indicates the precursor,
proHNP.