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. Author manuscript; available in PMC: 2017 Feb 9.

Published in final edited form as: Cell Signal. 2010 Jun 4;22(10):1502–1512. doi: 10.1016/j.cellsig.2010.05.019

Fig. 3 — Characterization of recombinant, expressed MK3.1-V5 and MK3.2-V5. (A) Subcellular localization of MK3.1-V5 or MK3.2-V5 in response to osmotic stress. HEK293t cells were transfected with pIRES-MK3.1-V5-EGFP or pIRES-MK3.2-V5-EGFP, starved, stimulated for 15 min with 0.3 M of sorbitol, fixed, labeled with V5 antisera and visualized by confocal fluorescence microscopy. Scale bar is 10 μM. (B) Osmotic stress induces a rapid breakdown of MK3.2-V5. HEK293t cells were transfected with pIRES-EGFP, pIRES-MK3.1-V5-EGFP or pIRES-MK3.2-V5-EGFP and, where indicated, were starved, stimulated for 15 min with 0.3Mof sorbitol, rinsed with cold TBS, lysed, and V5 immunoreactivity revealed following SDS-PAGE and immunoblotting. Where indicated, cells were pretreated with SB203580 (10 μM) for 10 min and then maintained for an additional 15 min in the presence of SB203580 and in the presence or absence of 0.3M sorbitol. Membranes were then stripped using 0.2 M NaOH (2×10 min) and reprobed using EGFP-specific antisera. In separate immunoblots, employing the same samples, filters were probed for phosphorylated p38 MAPK, stripped, and reprobed for total p38α immunoreactivity. Numbers at the left indicate the positions of the molecular mass marker proteins (in kDa). (C) Osmotic stress induces the phosphorylation of MK3. HEK293t cells were transfected with pIRES-EGFP, pIRES-MK2-V5-EGFP, pIRES-MK3.1-V5-EGFP or pIRES-MK3.2-V5-EGFP and, where indicated, were starved, stimulated for 15 min with 0.3 Mof sorbitol, rinsed with cold TBS, lysed, and probed with an antibody against human MK2phospho-threonine-222 following SDS-PAGE and immunoblotting. MK3.1-V5 and MK3.2-V5 were immunoprecipitated from lysates (1 mg) using an anti-V5 antibody. (D) MK3.2-V5T203A does not degrade in response to osmotic stress. HEK293t cells were transfected with pIRES-EGFP, pIRES-MK3.2-V5-EGFP or pIRES-MK3.2-V5 T203A-EGFP and, where indicated, were treated as described for panel B. (E) Effect of leptomycin B and MG132 upon the stability of MK3.2 during osmotic stress. HEK293t cells were transfected with pIRES-MK3.1-V5-EGFP or pIRES-MK3.2-V5-EGFP and, where indicated, were starved, stimulated for 15 min with 0.3 M of sorbitol, rinsed with cold TBS, lysed, and V5 immunoreactivity revealed following SDS-PAGE and immunoblotting. Where indicated, cells were pretreated with leptomycin B (LMB; 20 nM, 30 min) [21] or MG132 (50 μM, 4 h) [59] and then maintained for an additional 15 min in the presence of LMB or MG132 and in the presence or absence of 0.3 M sorbitol. Cells were then rinsed with cold TBS, lysed, and V5 immunoreactivity revealed following SDS-PAGE and immunoblotting.

Fig. 3 — Characterization of recombinant, expressed MK3.1-V5 and MK3.2-V5. (A) Subcellular localization of MK3.1-V5 or MK3.2-V5 in response to osmotic stress. HEK293t cells were transfected with pIRES-MK3.1-V5-EGFP or pIRES-MK3.2-V5-EGFP, starved, stimulated for 15 min with 0.3 M of sorbitol, fixed, labeled with V5 antisera and visualized by confocal fluorescence microscopy. Scale bar is 10 μM. (B) Osmotic stress induces a rapid breakdown of MK3.2-V5. HEK293t cells were transfected with pIRES-EGFP, pIRES-MK3.1-V5-EGFP or pIRES-MK3.2-V5-EGFP and, where indicated, were starved, stimulated for 15 min with 0.3Mof sorbitol, rinsed with cold TBS, lysed, and V5 immunoreactivity revealed following SDS-PAGE and immunoblotting. Where indicated, cells were pretreated with SB203580 (10 μM) for 10 min and then maintained for an additional 15 min in the presence of SB203580 and in the presence or absence of 0.3M sorbitol. Membranes were then stripped using 0.2 M NaOH (2×10 min) and reprobed using EGFP-specific antisera. In separate immunoblots, employing the same samples, filters were probed for phosphorylated p38 MAPK, stripped, and reprobed for total p38α immunoreactivity. Numbers at the left indicate the positions of the molecular mass marker proteins (in kDa). (C) Osmotic stress induces the phosphorylation of MK3. HEK293t cells were transfected with pIRES-EGFP, pIRES-MK2-V5-EGFP, pIRES-MK3.1-V5-EGFP or pIRES-MK3.2-V5-EGFP and, where indicated, were starved, stimulated for 15 min with 0.3 Mof sorbitol, rinsed with cold TBS, lysed, and probed with an antibody against human MK2phospho-threonine-222 following SDS-PAGE and immunoblotting. MK3.1-V5 and MK3.2-V5 were immunoprecipitated from lysates (1 mg) using an anti-V5 antibody. (D) MK3.2-V5T203A does not degrade in response to osmotic stress. HEK293t cells were transfected with pIRES-EGFP, pIRES-MK3.2-V5-EGFP or pIRES-MK3.2-V5 T203A-EGFP and, where indicated, were treated as described for panel B. (E) Effect of leptomycin B and MG132 upon the stability of MK3.2 during osmotic stress. HEK293t cells were transfected with pIRES-MK3.1-V5-EGFP or pIRES-MK3.2-V5-EGFP and, where indicated, were starved, stimulated for 15 min with 0.3 M of sorbitol, rinsed with cold TBS, lysed, and V5 immunoreactivity revealed following SDS-PAGE and immunoblotting. Where indicated, cells were pretreated with leptomycin B (LMB; 20 nM, 30 min) [21] or MG132 (50 μM, 4 h) [59] and then maintained for an additional 15 min in the presence of LMB or MG132 and in the presence or absence of 0.3 M sorbitol. Cells were then rinsed with cold TBS, lysed, and V5 immunoreactivity revealed following SDS-PAGE and immunoblotting.