Establishment of an in vitro Stat1 PTPase assay. (A) Bacterially produced p-Stat1 and Stat1 (∼8 ng) were analyzed by Western blotting with anti-Stat1 (top) or anti-p-Stat1(Y701) antibody (bottom). An extract from IFN-γ-stimulated HeLa cells (50 μg) was included as a control. (B) Bacterially produced p-Stat1 and an extract from IFN-γ-stimulated K562 cells were analyzed by gel mobility shift assay with a Stat1 binding sequence. The arrow indicates the Stat1 homodimer-DNA complex. (C) In vitro Stat1 PTPase assay. Bacterially produced p-Stat1 and 2.5 μl of HeLa cell nuclear extract (ne) were incubated at 30°C for various periods as indicated. The reaction mixtures were analyzed by Western blotting with anti-p-Stat1(Y701) antibody. (D) In vitro Stat1 PTPase assay performed as in panel C with inclusion of 0.5 mM sodium orthovanadate (OV) as indicated.

Purification and identification of a nuclear Stat1 phosphatase. (A) Schematic diagram of the chromatographic columns used to purify the Stat1 phosphatase activity. (B) Analysis of MonoS fractions. (Top panel) Western blot analysis of reaction mixtures from in vitro PTPase assays by using anti-p-Stat1 antibody. The samples analyzed include MonoS fractions S40 through S52 and the pooled fractions from the Resource Phenyl column (R33-41). —, p-Stat1 input. (Middle panel) Silver-staining analysis of the same MonoS fractions (15 μl/lane). A band of about 45 kDa (the presence of which correlates with the PTPase activity detected, as shown in the upper panel) is indicated by an arrow. (Bottom panel) Analysis of the same MonoS fractions (2.5 μl/lane) by Western blotting with the TC-PTP monoclonal antibody CF4. (C) Western blot analysis of 45 μg of HeLa whole-cell extract (wce), 45 μg of nuclear extract (ne), and 1 μl of MonoS fraction 46 (S46) with anti-TC-PTP antibody. (D) Analysis of TC45-immunodepleted HeLa nuclear extract. (Top panel) Anti-TC-PTP Western blot of HeLa nuclear extracts depleted with normal mouse IgG (ne-IgG) or the anti-TCPTP monoclonal CF4 (ne-TCPTP). (Bottom panels) Western blot analysis of a PTP assay performed with the immunodepleted nuclear extracts. The anti-p-Stat1 blot was stripped and reprobed with a Stat1 antibody.

TC45 can dephosphorylate Stat1 in vitro and in vivo. (A) Purified GST-TC45 dephosphorylates p-Stat1 in vitro. p-STAT1 was incubated with bacterially produced GST or GST-TC45, in the absence or presence of 0.5 mM orthovanadate (OV), and analyzed by Western blotting with anti-p-Stat1 or anti-Stat1 antibody. (B) TC45 dephosphorylates Stat1 in transiently transfected 293T cells. 293T cells transfected with pCMV-FlagStat1 (0.5 μg) and increasing amounts of pEBB-TC45 (10 to 200 ng) were stimulated 48 h posttransfection with IFN-γ for 20 min. Protein extracts were analyzed by Western blotting with anti-p-Stat1, anti-Flag, or anti-TC-PTP antibody as indicated. (C) Reduced IFN-γ-induced Stat1 phosphorylation in an U2OS cell line stably overexpressing TC45. Parental U2OS cells and U2OS-TC45 cells were stimulated with IFN-γ for various time periods. Protein extracts were analyzed as in panel B.

Analysis of Stat1 signaling in wild-type and TC-PTP-null MEFs. (A) Wild-type 7^+/+ and TC-PTP-null 4^−/− MEFs were stimulated with murine IFN-γ for various periods. Whole-cell lysates were analyzed by Western blotting with anti-p-Stat1 antibody. The stripped blots were reprobed with an anti-Stat1 antibody detecting both Stat1α and Stat1β. (B) 7^+/+ and 4^−/− MEFs were stimulated with IFN-γ for various periods with the addition of 0.5 μM staurosporine (Sigma) after 10 min of IFN-γ treatment. Whole-cell lysates were analyzed as in panel A. (C) 7^+/+ and 4^−/− MEF cells were stimulated with IFN-γ for 30 min, followed by a staurosporine chase (0.25 μM) for another 30 or 60 min. Cytoplasmic and nuclear extracts were analyzed by Western blotting with anti-p-Stat1 and antiactin antibodies, as indicated. Similar experiments were also performed with a different pair of wild-type and TC-PTP-null MEF cell lines (11^+/+ and 14^−/−). (D) p-Stat1 was incubated with dialyzed nuclear extracts of unstimulated 7^+/+ and 4^−/− cells in this in vitro PTPase assay and analyzed by anti-p-Stat1 Western blotting. The stripped blot was reprobed with a Stat1 antibody. —, dialysis buffer.

Reconstitution of TC-PTP-null MEFs with TC45, but not TC48, rescues the defect in Stat1 dephosphorylation. (A) Whole-cell extracts (75 μg/lane) from 7^+/+, 4^−/−, and 4^−TC45 cells were analyzed by Western blotting with the 3E2 monoclonal anti-TC-PTP antibody. (B) 7^+/+, 4^−/−, 4^−C, and 4^−TC45 cell lines were stimulated with IFN-γ for 30 min followed by a staurosporine chase (0.25 μM) for various periods as indicated. Whole-cell extracts were analyzed by Western blotting with the p-Stat1 antibody. Stripped blots were reprobed with anti-Stat1 and antiactin antibodies as indicated. (C) Puromycin-resistant pools of 14^−/− cells retrovirally infected with pBabepuroFlag-TC45 (14^−TC45∗) or pBabepuroFlag-TC48 (14^−TC48∗) were analyzed for expression with a Flag antibody. (D) Staurosporine-chase experiment as described above with the 14^−TC45∗ and 14^−TC48∗ pools.

Analysis of the dephosphorylation of Stat3, Stat5, and Stat6 in wild-type and TC-PTP-null MEFs. (A) 7^+/+ and 4^−/− MEFs were treated with 10 ng of murine IL-6 per ml (Peprotech) for 15 min, followed by a staurosporine chase for various periods as indicated. Whole-cell extracts were analyzed by Western blotting with anti-p-Stat3(Y705) antibody. The stripped blot was reprobed with anti-Stat3 antibody. (B) 7^+/+ and 4^−/− cells were stimulated with 500 ng of hGH per ml (Sigma) for 30 min followed by a staurosporine chase. Whole-cell extracts were analyzed with anti-p-Stat5(Y694) and anti-Stat5 antibodies. (C) 7^+/+ and 4^−/− cells were treated with 10 ng of murine IL-4 per ml (Peprotech) for 30 min followed by a staurosporine chase. Whole-cell extracts were analyzed with anti-p-Stat6(Y641) and anti-Stat6 antibodies.

Analysis of Stat1 and Stat5 dephosphorylation in wild-type and TC-PTP-null thymocytes. (A) Primary thymocytes isolated from wild-type and TC-PTP^−/− mice were treated for 15 min with 500 U of murine IFN-α (Calbiochem) and 80 U of IL-2 per ml (Invitrogen), respectively, followed by a staurosporine chase. Whole-cell extracts were analyzed with anti-p-Stat1 and anti-p-Stat5 antibodies, respectively. The stripped blots were reprobed with anti-Stat1 and anti-Stat5 antibodies. (B) Wild-type thymocytes were treated with IL-2 with or without addition of staurosporine to demonstrate the effect of staurosporine on Stat5 phosphorylation. Samples were analyzed as in panel A.