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. Author manuscript; available in PMC: 2013 Mar 7.
Published in final edited form as: Methods Mol Biol. 2011;720:279–292. doi: 10.1007/978-1-61779-034-8_17

Figure 1. mRNP assay of Ras12V cell lysate to measure endogenous RBP binding to ODC RNA.

Figure 1

To determine endogenous binding of HuR to the ODC transcript, HuR was immunoprecipitated from Ras12V cells as described in the Methods. Cell lysates isolated in RLB were incubated with a 50% (v/v) suspension of Protein A-sepharose beads precoated with either IgG1 or anti-HuR (Santa Cruz Biotech). After washing in NP40-based buffer, beads were treated with RNase-free DNase I and proteinase K, then RNA was extracted and reverse transcribed to obtain cDNA. The presence of ODC was analyzed by conventional PCR using primer pairs corresponding to a 635 bp region within the ODC transcript (see Note 8). Lanes labeled “Input RNA” represent duplicate PCR reactions performed with the same ODC primers on cDNA prepared from cell lysate without immunoprecipitation, indicating the presence of ODC RNA in the lysate.