Figure 3. Interaction of HA-DDX3X with PABP1 or caprin-1 is RNA-dependent.

(A) Cell lysates were prepared from spreading cells expressing HA-DDX3X; aliquots containing 500 µg of total protein were incubated with anti-HA-Agarose resin, in the presence or absence of RNAse1, to recover HA-DDX3X and associated proteins. Total extract (5% of input; lane 1) or recovered proteins (lanes 2 and 3) were separated by SDS–PAGE and visualised using immunoblotting using the antisera shown. (B) The soluble fraction of confluent cells (lane 1; 5% input) was incubated with m⁷GTP-Agarose to isolate eIF4E and associated proteins (lane 2), or with Agarose resin alone (lane 3). Protiens which co-isolated with eIF4E were separated by SDS–PAGE and visualised using immunoblotting. (C) Cell lysates were prepared from spreading cells; aliquots containing 500 µg of total protein were incubated with antibody specific to PABP1 followed by Protein A-Sepharose to recover PABP1 and associated proteins. Lysates were incubated with or without antibody, in the presence or absence of RNAse1, as described in the Materials and Methods section. Total protein and recovered proteins were separated by SDS–PAGE and visualised using immunoblotting using the antisera shown. Total extract (5% of input; lane 1) recovered proteins (lanes 2–4) and incubated with antibody (lanes 2 and 3) and resin only (lane 4).