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. Author manuscript; available in PMC: 2011 Jun 6.
Published in final edited form as: J Immunol. 2010 Oct 1;185(9):5198–5204. doi: 10.4049/jimmunol.1001224

FIGURE 2.

FIGURE 2

HuR binds to the 3′-UTR of cyclin D3 mRNA only in T cells cultured in the presence L-Arg. A, RNA-binding shift assay, where cell extracts from T cells cultured in medium with and without L-Arg for 24 and 48 h were mixed with an in vitro-transcribed radiolabeled 3′-UTR cyclin D3 mRNA. Specificity of the reaction was determined by adding an excess (15 or 150 pg) of unlabeled in vitro-transcribed cyclin D3 3′-UTR mRNA (related) or GAPDH mRNA (unrelated) to the protein extracts of activated T cells cultured with L-Arg and radiolabeled 3′-UTR cyclin D3 mRNA. B, Expression of HuR, AUF1, and TTP in whole-cell extracts from T cells cultured for 48 h in medium with or without L-Arg by Western blot. C, Blocking Abs against TTP and HuR were added before the mix with the in vitro-transcribed mRNA. D, Activated T cells were cultured in the presence or the absence of L-Arg for 48 h, after which mRNA was isolated from cells (input), cytoplasmic protein extracts (Cyto), and after immunoprecipitation of HuR or TTP. The mRNA was then tested for the expression of cyclin D3 mRNA using RT-PCR. Values are from three similar experiments.