Fig 2: The binding of rat Anxs to total RNA, total poly(A)-containing mRNAs and specific RNAs. Panel (A) shows schematically the protocol for the spotting of proteins on membranes to ease the evaluation of the data. Recombinant rat AnxA1-to-AnxA11, AnxA13 and BSA were spotted on nitrocellulose membranes in 3 µL samples containing 5, 10 or 20 µM protein, as indicated in Panel (A). Subsequently, the membranes were incubated for 30 min with 200,000 cpm of the following probes: [32P]-labeled total RNA [Panel (B)] or [32P]-labeled poly(A) -containing total mRNA [Panel (C)], in vitro transcribed [32P] rUTP-labeled anxA2 mRNA Panel (D), c-myc 3'UTR [Panel (E)], or the coding region of hRLuc Panel (F)]. All incubations were carried out in RNA-binding buffer in the presence of 100 µM Ca2+ and 1 µg/µL tRNA to block unspecific binding of RNA. Heat-denatured AnxA2 and BSA were spotted as negative controls since they do not bind RNA. The radioactive signals were detected after 48 h exposure of the spot blots to a radiosensitive screen in a phosphor-imager (Fuji Bas-5000). The dark signals (spots) on the membrane indicate RNA binding to the indicated recombinant rat Anx proteins. Panel (G) Samples of Anxs as indicated were subjected to 4%–15% SDS-PAGE and the proteins were stained with Coomassie Brilliant Blue. Standard proteins (PageRuler prestained protein ladder) are indicated to the left. The spot blots are representative blots from 3 experiments.