Figure 3

Phosphatase activities and PP2A methylation in adult mouse brain slices treated with recombinant SET protein for 5 h 30 min. A) The release of p-nitrophenol (nmoles of p-nitrophenol/mg of protein) was assessed. Results are expressed as a percentage of control (Ctrl) activity that was set to 100%. Mean ± SEM of 11 independent experiments; okadaic acid (OA); OA versus Ctrl: p < 0.0001; SET versus Ctrl: p = 0.0008. B) Release of p-nitrophenol measured on immunoprecipitated PP2A. Mean ± SEM of three independent experiments; OA versus Ctrl: p = 0.0025; SET versus Ctrl: p = 0.0124. C) Representative western blot (40 μg of protein loaded) of total and methylated PP2A (methyl-PP2A) and quantification of the ratio of methylated PP2A to total PP2A D). Mean ± SEM of nine independent experiments. SET versus Ctrl: p = 0.0057. E) Representative western blot of total PP2A and methyl-PP2A co-immunoprecipitated with a SET primary antibody (out of three independent experiments). Mouse brain slices in the absence (-) or the presence of recombinant SET protein (+) were immunoprecipitated with a SET antibody. Co-immunoprecipitated proteins were analyzed by western blotting, with a total PP2A antibody (lanes 3–6) and with a methylated PP2A (methyl-PP2A) antibody after membrane stripping (lanes 9–12). PP2A (lanes 3 and 5) and methyl-PP2A (lanes 9 and 11) are not detectable in absence of primary SET antibody (IP Ctrl). Similar levels of total PP2A (lanes 1 and 2) and methyl-PP2A (lanes 7 and 8) in total extracts before SET immunoprecipitation. F) Quantification of the ratio of methyl-PP2A to total PP2A co-immunoprecipitated with SET primary antibody in mouse brain slices treated with recombinant SET protein for 5 h 30 min and control (Ctrl) brain slices (no internalization). Meam ± SEM of three independent experiments; SET versus Ctrl: p = 0.0027.