Figure 3.

Identification of the RNA-binding proteins interacted with the gastrin mRNA. (A) Biotin-labeled probes A, B, and C were incubated with cytosolic extracts and pulled down by streptavidin beads. The mixtures were analyzed by SDS-PAGE combined with silver staining. (B) hnRNP K and PCBP1 were identified as major binding proteins interacted with gastrin mRNA 3′UTR by mass spectrometry analysis. (C) hnRNP K and PCBP1 interacted with probes B and C in vitro. The UV-cross-linking/biotin pull-down analysis and Western blot were performed with hnRNP K and PCBP1 antibodies. Beads represent the streptavidin beads control. (D) hnRNP K and PCBP1 bound to gastrin mRNA in vivo. AGS cells were treated with or without 10 nM EGF for 1 and 4 h, and then the cytoplasm proteins were harvested and incubated with anti-hnRNP K, anti-PCBP1, or anti-actin antibodies. RNA from immunoprecipitated complex was extracted, and the mRNA expression levels of gastrin and actin were measured by RT-PCR analysis (M, marker; P, positive control; N, negative control).