Fig. 4.

Confirmation of the presence of AnxA2 in Δ4 RNA–protein complex. (A) RNA gel mobility supershift analysis. CC polysomes from ethanol-exposed mice were incubated with ³²P-labeled Δ4 sense RNA and then increasing concentrations of either anti-AnxA2 (lane 2, 0.1 μL; lane 3, 0.2 μL; lane 4, 0.3 μL; lane 5, 0.4 μL; lane 6, 0.5 μL) or normal rabbit serum (NRS) (lane 7, 0.3 μL; lane 8, 0.4 μL; lane 9, 0.5 μL) were added. Samples were incubated for an additional 20 min and processed as for RNA gel shift analysis. A representative autoradiogram is shown here. A supershift (arrow on the right) was observed upon addition of anti-AnxA2 (lanes 2-6) but not with normal rabbit serum (lanes 7-9). Free probe and Δ4 RNA–protein complex are indicated on the left. Addition of anti-AnxA2 or normal rabbit serum to the incubation mix is indicated by (+) below the lanes, whereas an increase in the concentration of antibody or normal rabbit serum is indicated by the open triangles above the gel. Experiments were repeated three times with similar results. (B) RNA gel mobility assays. Purified recombinant AnxA2 protein or CC polysomes from control mice were incubated with [³²P]-labeled Δ4 sense RNA and samples were processed as detailed in Materials and methods. A shift of Δ4 RNA was observed upon incubation of RNA with CC polysomes (bracket on the left) or full-length recombinant AnxA2 protein (open arrow on the right). Arrow on the left indicates the position of free probe. Note that gel lanes between lane CC and lane AnxA2 have been omitted. Experiments were repeated three times with similar results. (C) Specificity of AnxA2 binding to Δ4 RNA. CC polysomes from control mice (lane 1), increasing concentration of purified recombinant thyroglobulin (lane 2, 0.25 μg protein; lane 3, 0.5 μg protein; lane 4, 1.0 μg protein) and bovine serum albumin (lane 5, 1.0 μg protein) were independently incubated with [³²P]-labeled Δ4 sense RNA and samples were processed as detailed in Materials and methods. A shift of Δ4 RNA was observed upon incubation of RNA with CC polysomes (bracket on the left) but not with thyroglobulin or bovine serum albumin. Arrow on the left indicates the position of free probe. Experiments were repeated three times with similar results.