Journal: Journal of Biological Chemistry

Article Title: Regulation of Dual-specificity Phosphatases M3/6 and hVH5 by Phorbol Esters

doi: 10.1074/jbc.m004182200

Figure Lengend Snippet: FIG. 1. hVH5 RNA transcript accumulation in response to PMA or anisomycin in K562 cells. A, top panel: time course of hVH5 transcript accumulation in K562 cells in response to PMA. PMA (200 nM) was added at 0 h. Only the smaller hVH5 transcript (2.5 kb) is shown. Bottom panel: 28 S RNA loading control. B, hVH5 transcript accumulation in response to anisomycin. Anisomycin (20 mg/ml) was added at 0 h. Bottom panel: the blot was reprobed with b actin cDNA as a loading control. C, effect of inhibitors of MEK1/2 (PD98059) or p38 (SB202474) on PMA-induced hVH5 transcript accumulation. K562 cells were pretreated with 100 mM SB202474 for 1 h or 30 mM PD98059 for 30 min before addition of PMA (200 nM). Cells were harvested 4 h after PMA addition. Bottom panel: the blot was reprobed with b actin cDNA as a loading control.

Article Snippet: For analysis of inducible M3/6 (myc) pro- tein, K562 cells (107) were metabolically labeled in the absence or presence of 2 mM Cd21 for 4 h in methionine or phosphate-free Dulbec- co’s modified Eagle’s medium containing 10% dialyzed bovine calf se- rum and 0.1 mCi/ml Tran35S-label (PerkinElmer Life Sciences) or car- rier-free 32PO4 (PerkinElmer Life Sciences).

Techniques: Control

Journal: Journal of Biological Chemistry

Article Title: Regulation of Dual-specificity Phosphatases M3/6 and hVH5 by Phorbol Esters

doi: 10.1074/jbc.m004182200

Figure Lengend Snippet: FIG. 2. Substrate specificity of M3/6 in K562 cells. Equal num- bers (106) of K562 cells stably transfected with the hMTIIa-M3/6 con- struct were treated with various concentrations of Cd21 for 4.5 h. Cells were then challenged with 200 nM PMA for 10 min, chilled on ice, and processed for Western analysis. A, M3/6 expression as revealed by staining with the anti-myc antibody 9E10(UCCC). B, the same blot was stripped and reprobed with antibody specific for phosphorylated SAPK; arrow indicates p46 SAPK. C, the blot was stripped and reprobed with antibody specific for phosphorylated ERK1/2, which are not resolved separately here. D, the blot was stripped and reprobed with antibody specific for phosphorylated p38.

Article Snippet: For analysis of inducible M3/6 (myc) pro- tein, K562 cells (107) were metabolically labeled in the absence or presence of 2 mM Cd21 for 4 h in methionine or phosphate-free Dulbec- co’s modified Eagle’s medium containing 10% dialyzed bovine calf se- rum and 0.1 mCi/ml Tran35S-label (PerkinElmer Life Sciences) or car- rier-free 32PO4 (PerkinElmer Life Sciences).

Techniques: Stable Transfection, Transfection, Western Blot, Expressing, Staining

Journal: Journal of Biological Chemistry

Article Title: Regulation of Dual-specificity Phosphatases M3/6 and hVH5 by Phorbol Esters

doi: 10.1074/jbc.m004182200

Figure Lengend Snippet: FIG. 3. Phosphorylation of M3/6 in K562 cells. A, equal numbers (106) of K562 cells stably transfected with the hMTIIa-M3/6 construct were treated with 2 mM Cd21 for 4 h. PMA (200 nM) was added for the indicated intervals, and the cells were processed for Western analysis using the anti-myc 9E10(UCCC) antibody. B, K562 cells stably trans- fected with the hMTIIa-M3/6 construct were treated with 2 mM CdCl2 for 5 h in the presence of 0.1 mCi/ml [32P]O4. 200 nM PMA (lane 3) or 20 mg/ml anisomycin (lane 4) was added for the last hour of incubation. Immunoprecipitates (9E10 anti-myc antibody) were subjected to elec- trophoresis followed by autoradiography. Arrows indicate positions of “unshifted” (lane 2) and mobility-shifted (lanes 3 and 4) material at the position of M3/6 protein.

Article Snippet: For analysis of inducible M3/6 (myc) pro- tein, K562 cells (107) were metabolically labeled in the absence or presence of 2 mM Cd21 for 4 h in methionine or phosphate-free Dulbec- co’s modified Eagle’s medium containing 10% dialyzed bovine calf se- rum and 0.1 mCi/ml Tran35S-label (PerkinElmer Life Sciences) or car- rier-free 32PO4 (PerkinElmer Life Sciences).

Techniques: Phospho-proteomics, Stable Transfection, Transfection, Construct, Western Blot, Incubation, Autoradiography

Journal: Journal of Biological Chemistry

Article Title: Regulation of Dual-specificity Phosphatases M3/6 and hVH5 by Phorbol Esters

doi: 10.1074/jbc.m004182200

Figure Lengend Snippet: FIG. 5. Half-life determination of M3/6 in K562 cells. A, K562 cells were incubated in methionine-free medium for 5 h in the presence of 2 mM CdCl2 and 0.1 mCi/ml [35S]methionine. PMA (200 nM) was added during the last hour of incubation. Aliquots were removed at the indicated times, lysates were prepared and immunoprecipitated with the 9E10 anti-myc antibody, and the washed immunoprecipitates were subjected to electrophoresis followed by autoradiography. B, densitometric analysis of the autoradiogram shown in A.

Article Snippet: For analysis of inducible M3/6 (myc) pro- tein, K562 cells (107) were metabolically labeled in the absence or presence of 2 mM Cd21 for 4 h in methionine or phosphate-free Dulbec- co’s modified Eagle’s medium containing 10% dialyzed bovine calf se- rum and 0.1 mCi/ml Tran35S-label (PerkinElmer Life Sciences) or car- rier-free 32PO4 (PerkinElmer Life Sciences).

Techniques: Incubation, Immunoprecipitation, Electrophoresis, Autoradiography