Fig. 3.

FMRP and NXF2 preferentially associate with Nxf1 mRNA-containing RNPs. FLAG-NXF2 was transfected into N2a cells, and IP was carried out 48 h after transfection. (A) Proteins from purified IP complexes or from 3% of supernatants were resolved on SDS/10% PAGE. The presence of the FMRP and NXF2 proteins in the IP complexes was confirmed by Western blot analyses. (Upper) IP with anti-FMRP (αFMRP) using mouse IgG as a negative control, followed by Western blotting using anti-FMRP. FMRP was in the anti-FMRP IP complexes (lane 4) but not in the IgG IP complexes (lane 3). (Lower) IP with anti-NXF2 (αNXF2) using preimmune serum as a negative control followed by Western blotting using anti-NXF2. NXF2 was in the anti-NXF2 IP complexes (lane 4) but not in the preimmune IP complexes (lane 3). Lanes 1 and 2 indicate that FMRP and NXF2 were present in the cell lysates. (B and C) RNAs were extracted from purified IP complexes followed by qRT-PCR. mRNA levels associated with FMRP (white bars) or NXF2 (gray bars) RNPs are indicated relative to those with negative control IP samples, which were arbitrarily set as 1. (B) A polyclonal anti-NXF2 antibody was used. (C) A monoclonal anti-FLAG antibody was used to immunoprecipitate the transfected FLAG-NXF2. Each bar represents mean ± SEM (B, n = 4; C, n = 2). (D) mRNA expression levels. RNAs were extracted from 10% of IP supernatants, and qRT-PCR was carried out using primers specific for each mRNA. Levels are plotted relative to Gapdh mRNA levels, which were arbitrarily set a value of 100. Each bar represents mean ± SEM (n = 4).