Fig. 2

PAF modulates 45 kDa and 48 kDa PTPN2 activity but not the 45 kDa PTPN2 localization. a Graph represents the phosphatase activity determined by the hydrolysis rate of pNPP by Flag-tagged PTPN2, immunoprecipitated from HEK-PAFR which had been starved overnight in DMEM 0.2% BSA before being stimulated with 100 nM PAF, for indicated times. After PTP experiments, proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted overnight with anti-PTPN2 antibodies for normalization of pNPP rates. Data are presented as low–high graphs (with lines at mean) of normalized pNPP hydrolysis rates for 3 independent experiments. Significance was established with paired two-way ANOVA with Sidak post-test. *p < 0.05. b, c HEK-PAFR, grown on poly-l-lysine coated coverslips, were transfected with Venus-tagged D182A-PTPN2 then stimulated, 30 h later, with vehicle or 100 nM PAF for 2, 5 or 10 min before permeabilization with 0.5% Triton X-100. Nuclei were stained with DAPI and cells were analyzed by confocal microscopy. Results are representative of 4 independent experiments, with at least 28 pictures analyzed from different fields per condition. Scale bar = 5 µm. b Compilation of Pearsons’ and Manders’ Coefficients for PTPN2. Data presented are mean ± SEM of 4 independent experiments and c representative pictures of Venus-tagged D182A 45 kDa or 48 kDa-D182A PTPN2 isoforms