Effect of PTPN7 on LPS-mediated MAPK signal transduction.

<p>(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.</p

Screening of PTPs regulated in RAW 264.7 cells in response to LPS.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078776#pone-0078776-t001" target="_blank">Table 1</a>. 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.</p

Inhibition of LPS-stimulated TNF-α production by PTPN7 in RAW 264.7 cells.

<p>(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. *<i>p</i><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. **<i>p</i><0.05 versus the LPS-treated control siRNA (Student’s t-test).</p

DUSP22-induced cell death.

<p>(A) Cell viability was determined as described in Materials and Methods. HEK 293 cells were transfected with FLAG-DUSP22 (WT or C88S mutant, 1 μg) expression plasmid. Data represent means ± SEM of six observations from three independent experiments. The expression levels of DUSP22 were determined by immunoblotting with an anti-FLAG antibody. IB, immunoblot. *<i>p</i> < 0.01 versus the control sample by Student’s <i>t</i>-test. (B) HCT 116 cells were transfected with 2 μg of FLAG-DUSP22 WT or FLAG-DUSP22 C88S expression plasmid. After 48 h of transfection, cell lysates were subjected to immunoblotting with indicated antibodies. Fifty microgram of cell lysate was loaded in each lane and the blot was probed with antibodies specific to p-JNK, cleaved PARP, or cleaved caspase-3. Twenty microgram of cell lysate was used for detection of JNK and FLAG. HCT 116 cells were transfected with (C) and (E) FLAG-DUSP22 WT (0, 0.5, 1, 2, 3, 4 μg) or (D) and (F) FLAG-DUSP22 C88S (0, 0.5, 1, 2, 3, 4 μg) for 48 h and then incubated for 1 h in the absence or presence of H₂O₂. Total cell lysates were subjected to immunoblot analyses with appropriate antibodies. Right panel: Relative fold of cleaved PARP levels after normalization to corresponding tubulin levels. All data are representative of three independent experiments.</p

Interaction of DUSP22 mutant with ASK1, MKK7, and JNK1 and dimerization of DUSP22.

<p>FLAG-DUSP22 WT or FlAG-DUSP22 C88S expression plasmid was co-transfected together with (A) HA-ASK1, (B) HA-MKK7, (C) HA-JNK1, or (D) HA-JNK2 expression plasmid. After 48 h, cells were lysed in lysis buffer and immunoprecipitated with the anti-FLAG M2 affinity gel. Pulled-down DUSP22 WT and mutant complexes were detected by immunoblotting with anti-HA and anti-FLAG antibodies. After 48 h, cells were lysed and immunoprecipitated with the anti-FLAG M2 affinity gel. The immunoprecipitates were subjected to SDS-PAGE and then immunoblotting with an anti-HA antibody.</p

Interaction of DUSP22 with ASK1, JNK1/2, and MKK7.

<p>(A) HEK 293 cells were co-transfected using FLAG-DUSP22 expression plasmid with or without HA-ASK1 expression plasmid. After 48 h, lysates were prepared and incubated with the anti-FLAG M2 affinity gel. Pulled-down DUSP22 complexes were subjected to SDS-PAGE. Immunoblot analysis was performed using an anti-HA antibody. IP, immunoprecipitation. (B) HEK 293 cells were co-transfected with HA-DUSP22 expression plasmid with or without FLAG-ASK1 expression plasmid. After immunoprecipitation, pulled-down ASK1 complex was detected as described above. HEK 293 cells were transfected with FLAG-DUSP22 expression plasmid together with (C) HA-ERK1, HA-JNK1, or HA-p38γ (D) HA-MKK4 or HA-MKK7. Cells were lysed in PTP lysis buffer and immunoprecipitated with the anti-FLAG M2 affinity gel. The immunoprecipitates were subjected to SDS-PAGE and then immunoblotting with anti-HA and anti-FLAG antibodies.</p