Immunoblot characterization of anti-RNS1 and
anti-RNS2 antibodies and RNS1 and RNS2 regulation in response to Pi
starvation. Protein samples were resolved by SDS-PAGE, transferred to a
membrane, and immunodetected with the corresponding antiserum. A,
Immunoblot with lanes containing approximately 70 ng of RNS1, RNS2, or
RNS3 in supernatant from RNase-expressing yeast cells as indicated or
30 μg of total proteins from Arabidopsis seedlings grown on media
rich (P+) or deficient (P−) in Pi. The blot was developed using
anti-RNS1 antibodies. B, Immunoblot with lanes containing approximately
300 ng of RNS1, RNS2, or RNS3, as indicated, from supernatant from
RNase-expressing yeast cells. Arab, Lane containing 50 μg of proteins
extracted from aboveground tissues of 5-week-old wild-type Arabidopsis
plants. The blot was developed using anti-RNS2 antibodies. C, Increase
in RNS2 abundance during Pi starvation. Lanes contain 25 μg of
protein extracts from seedlings grown on media rich (P+) or deficient
(P−) in Pi. The blot was developed using the same antibody as in B.
Positions of molecular mass markers (in kD) are shown to the left of
the blots.