(A) Dna2p and Tor1p do not interact in
vitro. Extracts from CB018 transformants were prepared and
immunoprecipitations (labeled “IP”) performed with either
affinity-purified anti-Dna2p antibodies (α-Dna2p) or 12CA5
(α-Tor1p) ascites. Washed immunoprecipitates were loaded onto 6%
SDS-PAGE gels and immunoblotted with either anti-Dna2p
(left) or anti-HA (right) antibodies. + and − indicate the
presence and absence, respectively, of extract in the
immunoprecipitate. (B). Effect of Tor1p overexpression on Dna2p levels.
Left panel, strain CB018 was transformed with either PRS315 (vector) or
pYDF125 (TOR1), and immunoblots were probed
with either affinity-purified anti-Dna2p antibodies or anti-actin
antisera. Right panel, strains YMW1(WT),
2–71(dna2–20), and 2–174(dna2–21)
were transformed with pYDF77
(GAL1p→TOR1) and grown for 5 h in
YPGal and either prepared for extracts (Gal) or shifted to YPD for
9 h (Dex) before preparation of extracts. Equal amounts of protein
were loaded onto 8% gels and immunoblots probed with
either anti-Dna2p (top) or anti-actin (bottom) antibodies. Protein
levels were quantified by densitometry and normalized to actin
levels.