(A) A schematic diagram of the FERM, PDZ, and catalytic domain (PTPase) of PTPN4. The targeting construct was designed with a loxP site between exon 20 and 21 followed by loxP sites flanking the neomycin cassette. Probe 7 was used in Southern blot analysis to detect the targeted locus and the KO locus. B, Bam HI; E, Eco RI; X, Xba I; neo, neomycin cassette. (B) Two ES cell clones (left panel: control, 3F5; positive for the targeted allele, 3F6) and F1 progeny from B6 crosses (right panel: lanes 1–3) were analyzed by Southern blotting to confirm the presence of the flanking loxP (floxed) targeted allele. Progeny from MORE-Cre crosses were examined for recombination of the floxed allele (wild type mice, +/+; heterozygotes, +/−; knock-out mice, −/−). Genomic DNA was digested with XbaI and hybridized with radiolabelled probe 7. Wild type (WT), targeted (flox), and recombined alleles are indicated. (C) To detect recombination of the targeted allele in mice, DNA was purified from either the tail or thymus of WT and PTPN4-deficient mice and amplified with PCR primers. (D) PTPN4 mRNA from the thymus of WT and PTPN4 knockout mice was amplified using RT-PCR for the full-length and the catalytic regions of PTPN4. (E) Equivalent amounts of messenger RNA, isolated from the thymus (T), spleen (S), and testes (Te) of WT (lanes 1–3) and PTPN4 knockout mice (lanes 4–6) were reversed transcribed into cDNA using primers for PTPN4 (upper panel, 2.7 kb) or GAPDH (lower panel, 0.3 kb). No product was detected when the reverse transcriptase was excluded from the reaction (lane 7).

(A) PTPN4 WT (+/+) and knockout (−/−) T cells were left untreated or stimulated for 10, 60, and 90 minutes with anti-CD3 mAbs. The cells were lysed and the lysates were Western immunoblotted with antibodies specific for phospho-ζ, phospho-Lck, phospho-ZAP70, phospho-Erk1/2, and GAPDH. (B) Total protein tyrosine phosphorylation was detected from whole cell lysates by Western blotting with anti-phosphotyrosine mAb (pY). Results are representative of three independent experiments.

WT and PTPN4 knockout mice were infected with LM intravenously. Three days later, the spleens (A) and livers (B) were removed. Homogenates from these organs were prepared, diluted, and aliquots then plated on brain-heart infusion medium plates. After an overnight culture, the number of bacterial colonies was determined/organ. Data are presented from six mice/group. The data are similar between two independent experiments.

(A) COS7 cells were co-transfected with Lck and TCR ζ. Cell lysates were subsequently prepared and mixed with GST-fusion proteins containing the catalytic domains of the indicated PTPases that were engineered as substrate-traps. Alternatively, TCR ζ was directly immunoprecipitated. The pull-downs and immunoprecipitates were resolved by SDS-PAGE and Western blotted with anti-phosphotyrosine (pY) followed by anti-TCR ζ. Data are representative of three independent experiments. (B) Recombinant PTPN4, purified from Sf9 insect cells, was incubated with purified, in vitro-phosphorylated ζ for the indicated time points. Two samples were incubated in the presence of 0.5 mM sodium molybdate (SM, lane 7) or sodium orthovanadate (SO, lane 8) for 30 min. The proteins from each sample were immunoblotted with phosphotyrosine-specific mAb. Data are representative of three independent experiments. (C) Lck and TCR ζ were co-transfected with a control vector (ctl, lane 1), full-length wild type (WT, lane 2) PTPN4, or a substrate-trapping mutant (DA, lane 3) of full-length PTPN4 into HEK 293T cells. After 48 h, the cells were lysed, and TCR ζ was directly immunoprecipitated from the cell lysates. The precipitates were Western blotted with anti-pY and anti-TCR ζ mAbs. Total cell extracts were also prepared and immunoblotted with anti-Lck mAbs. The insoluble fraction was immunoblotted with anti-PTPN4 and anti-β-actin mAbs. Data are representative of five independent experiments. (D) Jurkat T cells were co-transfected with 20 µg of NF-κB reporter construct and 2.5, 5.0, and 10 µg of WT or DA PTPN4. Total DNA was equalized with empty vector. Luciferase activity was measured in unstimulated and TCR/CD28-stimulated cells. Data are shown as mean ± SD (n = 3; **p < 0.01). Results are representative of three independent experiments. (E) AP-1 transcriptional activity was measured as described in (D). Luciferase activity was measured in unstimulated and TCR-stimulated cells. Data are shown as mean ± SD (n = 3; *p < 0.05; **p < 0.01). Results are representative of two independent experiments.

(A) Thymocytes isolated from WT (+/+) and KO (−/−) mice were stained with anti-CD4-PerCP 5.5 and anti-CD8-PE-Cy7 Abs. The double negative populations (CD4⁻CD8⁻B220⁻CD11b⁻) were further distinguished by staining cells with anti-CD44-APC and anti-CD25-FITC Ab. All cells were analyzed by flow cytometry. Data are representative of six experiments. (B) Gated double positive (DP) and CD4⁺ and CD8⁺ single positive populations from a representative WT mouse (gray solid line) or knockout mouse (black dashed line) were examined for CD3 and CD5 surface expression. Thymocytes were stained with anti-CD4-PerCP 5.5, anti-CD8-PE-Cy7, anti-CD3-Pacific Blue, and anti-CD5-PE Abs. An isotype control for CD3 or CD5 was used (light gray line). All cells were analyzed by flow cytometry. Data are represented as overlaid histograms and comparable to six independent experiments. (C) Lymph node cells of the indicated mice were isolated and stained with anti-CD3-Pacific Blue, anti-B220-APC-Cy7, anti-CD4-PerCP 5.5, and anti-CD8-PE-Cy7 Abs to examine peripheral lymphocytes populations by flow cytometry. Data are representative of six independent experiments. (D) Peripheral CD8⁺ and CD4⁺ T cells were stained with anti-CD62L-PE-Texas Red and anti-CD44-APC Abs to examine naïve and effector/memory subsets by flow cytometry. Data are shown as mean % ± SD (n = 4; *p = 0.003) and representative of three independent experiments.

(A) Purified T cells from a representative WT mouse (solid lines) or PTPN4 knockout mouse (dashed lines) were stimulated with anti-CD3/CD28 antibodies. Cells were harvested on Day 3 and stained with anti-CD4-APC, anti-CD8-PE-Cy7, anti-CD69 PE, and anti-CD25-FITC Abs to analyze upregulation of activation markers. Data are represented as overlaid histograms (light gray lines, unstimulated cells; black lines, stimulated cells). Results are representative of three independent experiments. (B) Purified T cells from a representative WT (solid lines) or PTPN4 knockout mouse (dashed lines) were labeled with CFSE and stimulated. Cell division was assessed by CFSE dilution by flow cytometry. Data are shown as overlaid histograms (light gray lines, unstimulated cells; black lines, stimulated cells). Data are representative of three independent experiments. Culture supernatants were collected from unstimulated and anti-CD3/CD28 stimulated T cells from WT (+/+) or PTPN4 knockout (−/−) mice (n = 6). (C) IL-2, IFN-γ, and TNF and (D) IL-4, IL-5, and IL-13 were measured from culture supernatants using the CBA assay.