Figure 4

GPKOW binds RNA in vivo. A. 293T cells were harvested and exposed to UV irradiation (254 nm/200 mJ). The cells were lysed and treated with T1 RNase (0.048 U/μl) for 8 min followed by immunoprecipitation with Dynabeads conjugated with anti-GPKOW1894 (+ anti-GPKOW, lane 2) or rabbit IgG (+ IgG, lane 1). Immunoprecipitated samples were dephosphorylated with calf intestinal phosphatase (0.038 U/μl). The RNA was labeled with γ³²-P ATP by PNK (0.5 U/μl) phosphorylation. All samples were separated on a denaturating (7 M urea) 6% PAA gel and subjected to autoradiography. The arrows indicate specific RNA species. The lower panel shows immunoreactive GPKOW from the cell lysate used (one representative lane is shown). B. Unsaturated images from the Typhoon 9410 phosphoimager (GE Healthcare Life Sciences) were quantified using the histogram function in Adobe Photoshop. The lanes were manually defined with identical frames. The statistical analysis was performed with paired students T-test in GraphPad Prism. The asterisk indicates a significant difference between the columns with a p-value < 0.05 (n = 4).