Figure 6

siRNA silencing of TRPM8 expression. Figure shows the level of TRPM8 immunoreactivity as determined by immunocytochemistry and by western blotting after treatment with siRNA oligonucleotides. (A) TRPM8 staining of a DRG growth cone treated with a non-specific control siRNA or with a specific TRPM8 siRNA oligonucleotide (#57381). The dotted line describes outline of the growth cone in the TRPM8 siRNA culture. (B) Quantitative analysis of TRPM8 immunocytochemistry shows that there was a significant decrease in TRPM8 expression in the presence of the TRPM8 siRNA (#57381, n = 73 growth cones) compared with the non-specific control siRNA (n = 56 growth cones). (C,D) Western blot and quantitation of TRPM8 immunoreactivity after treatment with 3 different TRPM8 siRNA oligonucleotides and a non-specific control siRNA. (C) Extracts from siRNA-treated DRG cultures were applied to SDS gels and probed with an anti-TRPM8 antibody. (D) Quantitative analysis of blots revealed significant knockdown of TRPM8 protein expression compared to cultures treated with a non-specific control siRNA. TRPM8 siRNA levels were normalised to GAPDH expression. Figure shows 60-70% knockdown achieved with 3 separate TRPM8 oligonucleotides. Values are means of 9 replicate immunoblot lanes over 3 separate experiments. Significant differences from control are depicted as: ** p < 0.005 as determined by a Mann-Whitney U-test. Error bars show means ± SEM