Figure 4

AIDA-1c associates with coilin in vivo and interacts directly with the C-terminus of coilin in vitro. (A) Lysates obtained from cells transfected with GFP-only, GFP-AIDA-1c or YFP-AIDA-1a [38] were immunoprecipitated with anti-GFP antibodies followed by Western analysis with anti-coilin antibodies (upper panel). Co-immunoprecipitated coilin can be observed most clearly in the AIDA-1c IP lane (lane 5). The blot was reprobed with anti-GFP antibodies to monitor the expression and immunoprecipitation of the GFP-tagged proteins (lower panel). The anti-GFP signal in lane 4 (bottom panel) has been underexposed six-fold relative to the signal in lanes 5 and 6. The expression level of GFP-AIDA-1c and YFP-AIDA-1a is low, resulting in no signal for these proteins in the input lanes (not shown). Input lanes account for 1/30 the amount of lysate used in the IP reactions. (B) AIDA-1c direct interaction with coilin is salt-sensitive. Purified coilin was incubated with GST-AIDA-1c in binding buffer containing various concentrations of NaCl. After the beads were washed, Western analysis was conducted using anti-coilin antibodies (upper panel). The blot was then stained with Ponceau S to verify that approximately equal amounts of GST-AIDA-1c were used in each reaction (lower panel). The input lane accounts for 20% of the protein used in the pulldown reactions. (C) AIDA-1c directly interacts with the C-terminus of coilin. Purified T7-tagged AIDA-1c was incubated with GST or GST-tagged coilin fragments containing either the C-terminal 156 or 214 amino acids of coilin. The blot was subsequently stained with Ponceau S to verify that approximately equal amounts of GST-fusions were used in the assay (lower panel). The input lane accounts for 20% of the protein used in the pulldown reactions. (D) AIDA-1c competes with Sm proteins for coilin binding sites. A competition experiment is shown wherein individual reactions were set-up containing a fixed amount of GST-coilin fragment (GST-C214) and T7-SmB' with increasing amounts T7-AIDA-1c as indicated. As can been seen in lane 6, as more AIDA-1c binds to GST-C214, less SmB' is recovered. Lane 2 shows the binding of either AIDA-1c (top) or SmB' (bottom) to GST-C214 in the absence of competitor. Input lanes account for 20% of the protein used in the 1X reactions.