Mapping the RHA binding domain of vigilin. (A) RHA binds to the KH10–12 region of vigilin. HEK 293 cells were cotransfected with Flag-tagged RHA and GFP-tagged vigilin mutants. Seventy-two hours after transfection, whole cell lysates were used for immunoprecipitation with Flag antibody conjugated beads. The presence of vigilin mutants in the precipitates was detected by Western bloting with anti-GFP antibody. Vigilin KH8–14 (lane 6) and KH10–14 (lane 10) regions, but not KH1–7 (lane 5) and KH13–14 (lane 14) regions associate with RHA. (B) The same samples were used for Western blotting with anti-flag antibody to confirm that the tagged constructs could be immunoprecipitated. (C) Association is independent of RNA. Experiments were carried out as in A except that cell lysates were treated with 200 μg/mL RNase A at 4°C for 2 h before immunoprecipitation. After RNase treatment, vigilin KH10–14 still associates with RHA (lane 4).
