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Fig. 4. Interaction of the RNA probes from the 3' UTR of the rat ₂ subunit of guanylyl cyclase with nuclear or cytosolic proteins extracted from granule cells. (A) A radiolabelled, in vitro transcribed RNA fragment corresponding to the 843 bp fragment obtained with F1 and R1 primers (see Fig. 3) was incubated with of cytosolic (Cyt) or nuclear (Nucl) protein extracts from control or NMDA-treated cells. After binding, RNA-protein complexes were treated with T1 RNase and resolved by non-denaturing gel electrophoresis as described in the Materials and Methods. Controls were performed without the protein extract (NP). (B) Radiolabelled, in vitro transcribed RNA fragments corresponding to that of described in panel A was incubated with nuclear (Nucl) protein extracts from NMDA-treated cells or control cells, in the presence or absence of the non-labelled probe (NLP). (C) Radiolabelled, in vitro transcribed RNA fragment corresponding to the 1176 bp fragment obtained with the F2 and R2 primers (see Fig. 3) was used in a similar experiment to that described in A. (D) The presence of endogenous ₂ mRNA was assayed in the IP material from cytosolic or nuclear extracts obtained with anti-HuR or anti-AUF1 by real-time RT-PCR. The results are the mean ± s.e.m. of two experiments. *P<0.01 and ⁺P<0.01, significant difference from control values (IgG).

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