Display Settings:

Items per page

Results: 6

1.
Figure 6.

Figure 6. From: Interphase-specific Phosphorylation-mediated Regulation of Tubulin Dimer Partitioning in Human Cells.

The significance of Op18 phosphorylation in response to T cell receptor/CD3 complex stimulation. (A) Jurkat T cells were stimulated as indicated with either the anti-CD3 antibody UCHT-1 (8 μg/ml) or the control anti-CD2 antibody OKT11 (8 μg/ml). MT polymer content was determined as in Figure 1 at the indicated time points. (B) Immunoblots of total proteins of Jurkat T cells expressing Vector-Co or shRNA-Op18-443, induced for 8 h to express either Op18-F or Op18-S16,25A-F. (C) The MT polymer content of interphase cells was determined without anti-CD3 stimulation (open bars) or with anti-CD3 stimulation (black bars, 8 μg/ml for 10 min) of the cells. The data are representative of three independent transfection experiments.

Per Holmfeldt, et al. Mol Biol Cell. 2007 May;18(5):1909-1917.
2.
Figure 3.

Figure 3. From: Interphase-specific Phosphorylation-mediated Regulation of Tubulin Dimer Partitioning in Human Cells.

The significance of Op18 and MAP4 for phosphorylation-mediated regulation of interphase MT polymers by CaMKIV and MARK2 kinases. K562 cells were transfected as in Figures 1 and 2 with the indicated combinations of shuttle vector shRNA and pMEP derivatives. (A) Immunoblots of total proteins of singly or doubly depleted cells induced for 8 h to express CaMKIV(c)-F. The MT polymer content of interphase cells was determined as in Figure 1. (B) MT regulation by the MARK2-HA kinase, shown using the same shRNA derivatives and conditions as in A. The data are representative of at least two independent transfection experiments.

Per Holmfeldt, et al. Mol Biol Cell. 2007 May;18(5):1909-1917.
3.
Figure 2.

Figure 2. From: Interphase-specific Phosphorylation-mediated Regulation of Tubulin Dimer Partitioning in Human Cells.

MT regulation in response to induced expression of protein kinases. K562 cells were transfected with the pMEP shuttle vector derivative indicated, counterselected with hygromycin for 5 d, and ectopic expression was induced for the indicated times. (A) Immunoblots of total proteins of cells induced to express the protein kinase indicated. (B) The MT polymer content of interphase cells described in A, determined as in Figure 1. (C) Immunoblots of total proteins from cells induced for 8 h to express CaMKIV(c)-F or MARK2-HA, alone or in combination. The histogram shows the MT polymer content of cells at interphase. (D) Mitosis-specific MT content, determined as in Figure 1. The data are representative of at least three independent transfection experiments.

Per Holmfeldt, et al. Mol Biol Cell. 2007 May;18(5):1909-1917.
4.
Figure 4.

Figure 4. From: Interphase-specific Phosphorylation-mediated Regulation of Tubulin Dimer Partitioning in Human Cells.

Importance of physiological Op18 levels in control of tubulin dimer partitioning. K562 cells were transfected as described in Materials and Methods with the indicated mixtures of EBV-based shuttle vector-Co, shRNA-Op18-443, and a Flag-tagged Op18 derivative (Op18-F), which was resistant to shRNA. Untransfected cells were counterselected with hygromycin for 5 d. (A) Immunoblot analysis of Op18 levels in cells transfected with the indicated shuttle vector derivative and induced to express ectopic Op18-F for the times indicated. Migration of endogenous Op18 (arrows) and Flag epitope-tagged Op18-F (arrowheads) is indicated. (B) The MT polymer content of interphase cells described in A was determined at the indicated time points, as in Figure 1. The data are representative of at least three independent transfection experiments.

Per Holmfeldt, et al. Mol Biol Cell. 2007 May;18(5):1909-1917.
5.
Figure 5.

Figure 5. From: Interphase-specific Phosphorylation-mediated Regulation of Tubulin Dimer Partitioning in Human Cells.

The significance of Op18 phosphorylation as evaluated by inducible gene product replacement. K562 cells were transfected as in Figure 1 with the indicated combinations of shuttle vectors. (A) Immunoblots of total proteins of control cells or cells expressing shRNA-Op18-443 induced for 8 h to express PKA and/or Flag-tagged Op18 derivatives either with the wild-type amino acid sequence (Op18-F) or with Ser-16 and Ser-63 substituted by Ala residues (Op18-S16,63A-F). The MT polymer content of interphase cells was determined after 8 h of induced expression as in Figure 1. (B) MT regulation by the CaMKIV(c)-F kinase is shown using the same shRNA and Op18 derivatives as in A. The data are representative of at least three independent transfection experiments.

Per Holmfeldt, et al. Mol Biol Cell. 2007 May;18(5):1909-1917.
6.
Figure 1.

Figure 1. From: Interphase-specific Phosphorylation-mediated Regulation of Tubulin Dimer Partitioning in Human Cells.

Interphase-specific opposing effects of Op18 and MAP4. K562 cells were transfected as described in Materials and Methods with the indicated mixtures of shuttle vector-Co, shRNA-Op18-443, or shRNA-MAP4-10. Untransfected cells were counterselected with hygromycin for 5 d. (A) Immunoblots of total lysates of transfected cells by using the indicated antibody for detection. Quantification was achieved by serial dilution, which revealed >96% specific depletion of Op18, ∼85% depletion of MAP4, but no alterations in tubulin levels. The PCNA protein was used as loading control. (B) Immunoblots of total protein from cells that expressed the indicated shRNA derivatives for 5 d. The MT polymer content of interphase cells was determined as described in Materials and Methods, and data are expressed as percentage of polymerized tubulin. (C) Total amount of polymerizable tubulin dimers in specifically depleted cells was determined by addition of Taxol for 2 h (2 μg/ml) followed by determination of MT polymer content by flow cytometry. Data are expressed as percentage of the MT polymer content of vector-Co cells at interphase. To confirm that Taxol promotes close to maximal polymerization under the present conditions, soluble tubulin dimers were analyzed by immunoblotting, which revealed that <3% was unpolymerized tubulin (data not shown). (D) Mitosis-specific MT polymer content of cells described in C was determined by flow cytometry, which allowed gating of mitotic cells as outlined under Materials and Methods (primary histograms are shown in Figure Supplement S2). Data are expressed as percentage of the MT polymer content of mitotic vector-Co cells. The data are representative of at least three independent transfection experiments.

Per Holmfeldt, et al. Mol Biol Cell. 2007 May;18(5):1909-1917.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk