Figure 5
Measurement of cellular PTPN2 activity in immunoprecipitates using FAM-pStat1 and RP-UFLC.
(A) Determination of the activity of immunoprecipitated endogenous PTPN2. Endogenous PTPN2 was immunoprecipitated from Jurkat cells extracts using an polyclonal anti-PTPN2 antibody (IP PTPN2). The immunobeads were incubated with FAM-pStat1 (50 μM final) for different time prior to RP-UFLC analysis. Beads not coated with anti-PTPN2 antibody were used as control (beads). The AUC of the dephosphorylated-FAM-pStat1 peptide peak was plotted versus time. Proteins bound to immunobeads (IP PTPN2) or control beads were also analyzed by Western blot using a monoclonal anti-PTPN2 antibody. (B) Immunoprecipitation studies with Jurkat cells and PTPN2-deficient human cells (SUP-HD1). Immunoprecipitations were carried out in parallel with extracts from Jurkat and SUP-HD1 cells using the polyclonal anti-PTPN2 antibody. The immunobeads were incubated with FAM-pStat1 (50 μM final) for RP-UFLC analysis. Proteins bound to immunobeads were analyzed by Western blot using the monoclonal anti-PTPN2 antibody. (C) Inhibition of endogenous PTPN2 by H2O2. Jurkat cells were left untreated (control) or treated 30 min with 20 μM H2O2. Endogenous PTPN2 was immunoprecipitated with a polyclonal anti-PTPN2 antibody and the immunobeads were incubated with FAM-pStat1 peptide (50 μM final) and the residual PTPN2 activity was determined (normalized to the control). *p < 0.05. Immunobeads-bound proteins from treated (H2O2) and not treated (control) were analyzed by Western blot using a monoclonal anti-PTPN2 antibody (PTPN2) or a monoclonal anti-oxydized PTP (after stripping of the membrane). *p < 0.05 versus non-treated cells (Mann-Whitney U test, Prisme software).
