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

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