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. 2012 Mar 19;3(3):299–313. doi: 10.18632/oncotarget.462

Figure 4. Identification and analysis of AREs motifs in BRCA1-IRIS 3`-UTR region.

Figure 4

(A) The 3`-UTR region of BRCA1-IRIS (i.e. part of BRCA1/p220 intron 11) where putative AUF-1 (red sequences) and pCBP2 (green sequences) binding sites are shown. (B) Plasmids carrying BRCA1-IRIS or c-fos 3`UTRs downstream of the luciferase gene in RSV-plasmid were transfecetd in HME with siGFP, siBRCA1/p220, siAUF1, sipCBP2 or siHuR and the luciferase activity expressed from these different plasmids was measured using luminometer. Data are represented as means ± SD from triplicates done three independent times (in all cases p≤0.01 compared to RSV-Luc alone). (C) Representative PCR analyses of the binding of BRCA1-IRIS mRNA to AUF1, pCBP2, HuR in untreated HME cells cytoplamsic proteins (polysomes), while binding only to HuR in BRCA1/p220-silenced HME cells. (D) Immunoprecipitation of the indicated proteins in BRCA1/p220-silenced HME cells (left), or from BRCA1/p220-reconstituted HCC1937 cells (right). (E) Immunoprecipitated mRNP-protein complex as percentage of input using AUF1, pCBP2, HuR and BRCA1/p220 antibodies and IgG from cells transfected with control or BRCA1/p220 siRNA. (F) Representative PCR analyses of the binding of AUF1 and pCBP2 to BRCA1-IRIS mRNA in BRCA1/p220-silenced HME cells (upper) or BRCA1/p220-overexpressing HCC1937 (lower) cytoplasmic proteins (polysomes).