Figure 6.

(A) Identification of native complexes assembled in SKBR-3 cells. The SKBR-3 cytoplasmic extracts were left untreated or treated with RNase cocktail on ice for 30 min. The extracts were immunoprecipitated with anti-HuR or anti-C1/C2 antibodies and the complexes immobilized on nitrocellulose. The presence of PABP, AUF, HuR, C1/C2, A1, and other hnRNP proteins was examined by immunodetection with corresponding antibodies. (B) Association of the endogenous Her-2 mRNA with the HuR/C1/C2 complexes in SKBR-3 cells. The RNA–protein complexes in SKBR-3 cells were cross-linked by formaldehyde and the extracts were made as described in Materials and Methods. The extracts were immunoprecipitated with either anti-HuR or anti-C1/C2 antibodies and the RNA–protein cross-links were reversed by heat treatment. The RNA associated with the complexes was extracted with Trizol and used for first-strand cDNA synthesis. The presence of Her-2 RNA or nonspecific GAPDH RNA was detected by PCR using gene-specific primers.