Figure 6

GPKOWs ability to bind RNA is sensitive to mutations of its PKA phosphorylation sites. A. U2OS cells were transfected with GPKOW wt (row 1), GPKOW S27A (row 2), GPKOW T316A (row 3) or GPKOW S27A+T316A (row 4). Cells were fixated with paraformaldehyde 20 hours later and subjected to immunofluorescence analysis using anti-GPKOW1894 (left, green). Nuclear DNA was visualized with Hoechst (middle, blue). Merged photos to the right. Scale bar 10 μM B. 293T cells transfected with GPKOW wt (lanes 1 and 2) or GPKOW S27A + T316A (lanes 3 and 4) were harvested and exposed to UV irradiation at 254 nm (200 mJ). The cells were lysed and immunoprecipitated with Dynabeads conjugated with either rabbit IgG (+ IgG, lanes 1 and 3) or anti-GPKOW1894 (+ anti-GPKOW, lanes 2 and 4). Immunoprecipitated samples were dephosphorylated with calf intestinal phosphatase (0.038 U/μl) followed by labeling of RNA with PNK (0.5 U/μl) phosphorylation. The samples were separated in a denaturating (7 M urea) 6% polyacrylamide 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). n = 4 C. 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 < 0.01 (n = 4).