FIG. 3.

Interaction of Srp1p and Sts1p as demonstrated by coimmunoprecipitation from cell extracts (A) and by the use of GST-Sts1p fusion protein (B). (A) Extracts were prepared from the strain carrying the HA-tagged STS1 gene (lanes 3 and 5) or the control strain without the HA-tag (lanes 2 and 4). The extracts were treated with anti-Srp1p antibodies (lanes 4 and 5) and protein A-Sepharose, and precipitated proteins were subjected to SDS-PAGE followed by Western analysis using a monoclonal anti-HA antibody to detect HA-Sts1p. Lane 1 is an antibody control without extracts. Lanes 2 and 3 are negative controls without antibody. (B) Srp1p was incubated with GST (lane 3), GST-Sts1p (lane 4), GST-Sts1ΔNLS1p (lane 5), GST-Sts1p plus 200 μM SV40 NLS peptide (lane 6), or GST-Sts1p plus 200 μM reverse NLS peptide (lane 7). Lane 2 did not receive any GST fusion protein. GST or GST fusion proteins were pulled down using glutathione-agarose beads and then subjected to SDS-PAGE followed by Western blot using anti-Srp1p antibodies to detect Srp1p. Lane 1 is 10% of the input Srp1p preparation.