FIG 10.
HuR RNP-IP assays of selected proinflammatory mRNAs. (A) qRT-PCR analysis of murine Pan02 cells transfected with a human HuR expression plasmid or empty vector using probes that detect human HuR. RNA extracted from human PDAC cell line Hs766T serves as positive control. (B) Validation of mRNP-IP performed with total and cytoplasmic fractions of Pan02 cells transfected with either HuR overexpression plasmid or vector control; α-tubulin was used as a loading control for the input and a negative control for the immunoprecipitation samples. Hs766T, Hs766TΔHuR (60), and a human normal pancreatic line, HPNE, serve as controls. (C) Quantification of total HuR protein relative to α-tubulin levels. (D) Relative binding of mRNA targets to HuR, normalized to respective IgG controls, determined by qRT-PCR using GAPDH mRNA as a loading control and Cox-2 as a positive control. *, P < 0.01; **, P < 0.001; n.s., not significant.