AUF-1 and YB-1 assemble a β-complex in vivo. (A) AUF-1 and YB-1 bind to the β-complex target motif. EMSA analyses were conducted using a [³²P]-labeled β-globin 3′UTR RNA in the presence of the GST-AUF-1(p40), GST-YB-1, control GST moiety, or K562 cytoplasmic extract. Select incubations were amended with sense (S) or antisense (AS) WT₁₅ competitor DNA. The positions of the AUF-1 and YB-1 mRNPs are indicated by an open arrow and the β-complex by a closed arrow, respectively. (B) Double knockdown of AUF-1 and YB-1 abolishes β-complex assembly in vivo. Top: EMSA analyses were conducted on a [³²P]-labeled β-globin 3′UTR RNA using cytoplasmic extracts prepared from siRNA-transfected K562β cells. An arrow indicates the β-complex. Bottom: K562β cells were transfected with specific siRNAs targeting AUF-1, YB-1, both AUF-1 and YB-1 (A/Y), or control lamin mRNAs. Cytoplasmic extracts were subsequently analyzed by immunoblot using factor-specific or control GAPDH Abs (ab).