FIG. 6.

Assembly of RFX complexes exhibiting DNA binding activity in transduced SJO cells. (A) RFX complexes are restored in SJO cells transduced with the bicistronic lentiviral constructs encoding wild-type (WT) and truncated (C2, C4, and C5) versions of RFX5. No RFX complex is detected in SJO cells transduced with a negative-control construct (stuffer). Binding of the RFX complexes was analyzed by EMSA with an oligonucleotide probe containing the X box of the DRA gene. The whole-cell extracts used for the experiments were prepared from the transduced cells after purification of these cells by sorting for CD8 expression. Only the region of the gel containing specific RFX-DNA complexes is shown. All lanes are from the same gel and at the same exposure. A weak nonspecific (ns) complex migrating just below the band due to wild-type RFX is indicated. (B) The mixtures of binding reactions performed as described above were supplemented with preimmune serum (PI), antibodies specific for the HA tag, or antibodies specific for the two other subunits of the RFX complex, RFXANK and RFXAP. The anti-HA antibody supershifts complexes containing the transduced wild-type, C2, C4, and C5 RFX5 proteins. The complex containing C5 is also supershifted by the antibodies directed against RFXAP and RFXANK.