PMC

(A) After PTPN7-transfected RAW 264.7 cells were stimulated with LPS (1 µg/ml) for 1 h, immunoblotting was performed for total and phosphorylated proteins as indicated. Relative phosphorylation levels of MAPKs were normalized to the expression levels of the corresponding total MAPKs and presented as fold increase. Data are representative of four independent experiments. (B) RAW 264.7 cells were transfected with control siRNA or PTPN7 siRNA #1 for 48 h, and were stimulated with LPS (1 µg/ml) for 1 h. MAPK activities were determined by immunoblotting with appropriate antibodies. Relative phosphorylation levels of MAPKs were normalized to the expression levels of the corresponding total MAPKs and presented as fold increase. Data are representative of four independent experiments.

Each cDNA was synthesized from total RNA that was extracted from RAW 264.7 cells at the indicated times after treatment with LPS (1 µg/ml). PTP transcripts were amplified using specific primers listed in . GAPDH transcripts were amplified using specific primers (forward 5′-ACCACCATGGAGAAGGC-3′; reverse 5′-CTCAGTGTAGCCCAGGATGC- 3′) as a control. Similar results were obtained in three independent experiments. The PCR products were visualized by ethidium bromide staining and quantified by scanning the gel images followed by analysis with LabWorks software (UVP Inc.). The PCR data were normalized to the expression level of GAPDH and are presented as relative fold changes.

(A) Secretion levels of LPS-stimulated TNF-α were increased time-dependently in cell culture supernatants. At different times post treatment with LPS, culture supernatants were analyzed for TNF-α production using ELISA assay. (B) RAW 264.7 cells were transfected with either the empty vector or the construct expressing FLAG-PTPN7. After 16 h stimulation with LPS (1 µg/ml), supernatants were analyzed for TNF-α production using an ELISA assay, as described under Materials and Methods. Cell lysates were subjected to immunoblotting using an anti-FLAG antibody for detection of PTPN7. The results presented represent the mean data from three independent experiments. *p<0.01 versus the vector controls (Student’s t-test). (C) PTPN7 knockdown (PTPN7 siRNA #1 and #2) was detected via immunoblotting using anti-PTPN7 and anti-tubulin antibodies. (D) After transfection with control or PTPN7 siRNAs (#1 and #2), cells were treated with LPS (1 µg/ml) for 1 h, and the levels of TNF-α were measured using an ELISA assay. The results presented represent the mean data from three independent experiments. **p<0.05 versus the LPS-treated control siRNA (Student’s t-test).

(A) PTPN7 transcript level was determined at the indicated times after LPS treatment (1 µg/ml) by quantitative real-time PCR in RAW 264.7 cells. GAPDH transcripts were amplified using specific primers as a control. Relative mRNA expression levels were normalized with GAPDH and presented as fold increase. The results presented represent the mean data from three independent experiments. *p<0.01 versus the untreated sample (Student’s t-test). (B) Expression of PTPN7 in RAW 264.7 cells were analyzed at various time points after LPS stimulation (1 µg/ml) by immunoblotting with appropriate antibodies. The experiments were repeated three times with similar results. Protein expression levels were quantified by scanning the immunoblots followed by analysis with LabWorks software (UVP Inc.). Relative expression levels of PTPN7 and DUSP1 were normalized to the expression level of tubulin and presented as fold increase. IB, immunoblot. (C) After RAW 264.7 cells were untreated or treated with 1 µg/ml of LPS for 2, 4, 6, 8, 16 h, cells were harvested and immunoprecipitated with an anti-PTPN7 antibody. In vitro phosphatase activity assays were performed as described in Materials and Methods. The results presented represent the mean data from three independent experiments. *p<0.01 versus the untreated sample (Student’s t-test). Protein expression levels were analyzed by immunoblotting and quantified by scanning the immunoblots followed by analysis with LabWorks software (UVP Inc.).