Journal:

Article Title: Assembly of cyclin D-dependent kinase and titration of p27 Kip1 regulated by mitogen-activated protein kinase kinase (MEK1)

doi:

Figure Lengend Snippet: Molecular correlates of S phase entry. (A) Cells from five individual subclones expressing inducible MEK1* were serum-starved and restimulated with ZnSO4 or FBS for 16 hr, and the S phase fraction was determined by using flow cytometry to estimate the DNA content of propidium iodide-stained nuclei. (B) Serum-starved cells (subclone 3) stimulated with ZnSO4 or FBS were assayed at 4-hr intervals for DNA content (Upper). Cyclin A, p21Cip1, and p27Kip1 levels were determined by immunoblotting. Precipitates recovered with antibodies to cyclin E and CDK2 were assayed for histone H1 kinase activity.

Article Snippet: Rabbit polyclonal antibodies against ERK1 (K-23), ERK2 (C-14), cyclin E (M-20), cyclin A (C-19), and p21 Cip1 (C-19) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Flow Cytometry, Staining, Western Blot, Activity Assay

Journal:

Article Title: Assembly of cyclin D-dependent kinase and titration of p27 Kip1 regulated by mitogen-activated protein kinase kinase (MEK1)

doi:

Figure Lengend Snippet: Overexpression of cyclin D1, CDK4, and MEK1* induces cyclin A synthesis and CDK2 activation, but not p27Kip1 degradation. Cells (subclone 2) ectopically expressing cyclin D1, CDK4, and inducible MEK1* were starved and restimulated with ZnSO4 or FBS. Cells harvested at intervals thereafter (hr) were assayed for cyclin A and p27Kip1 expression by immunoblotting (top two panels), for cyclin D1-associated p27Kip1 by immunoprecipitation with cyclin D1 followed by immunoblotting with anti-p27Kip1 (middle panel), and for cyclin E- and CDK2-associated histone H1 kinase activity (bottom two panels). More than 85% of p27Kip1 coprecipitated with cyclin D1.

Article Snippet: Rabbit polyclonal antibodies against ERK1 (K-23), ERK2 (C-14), cyclin E (M-20), cyclin A (C-19), and p21 Cip1 (C-19) were purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, Activation Assay, Expressing, Western Blot, Immunoprecipitation, Activity Assay

Journal:

Article Title: Apoptosis Induced by the Histone Deacetylase Inhibitor FR901228 in Human T-Cell Leukemia Virus Type 1-Infected T-Cell Lines and Primary Adult T-Cell Leukemia Cells

doi: 10.1128/JVI.78.9.4582-4590.2004

Figure Lengend Snippet: Expression of Bcl-2, Bcl-xL, Bax, and cyclin D2 in primary ATL cells treated with FR901228. Leukemic cells obtained from patients with ATL were treated with (+) or without (−) 5-ng/ml FR901228 for 24 h. Cell lysates (50 μg of protein per lane) were fractionated on SDS-polyacrylamide (10 or 12%) gels and analyzed by Western blotting with antibodies directed against each protein. Comparable protein loading was verified with an antibody specific for the housekeeping gene product actin.

Article Snippet: After blocking of the membranes in 3% skim milk and 0.05% Tween 20 in Tris-buffered saline, the blots were incubated with the mouse monoclonal antibody to Tax, Lt-4 ( 52 ), Bcl-2 (InnoGenex, San Ramos, Calif.), XIAP (Medical & Biological Laboratories Co., Nagoya, Japan), p53, p21, or Bax (NeoMarkers, Fremont, Calif.), or the rabbit polyclonal antibody to cyclin D2, actin (Santa Cruz Biotechnology), or Bcl-x L (Transduction Laboratories, Lexington, Ky.).

Techniques: Expressing, Western Blot

Journal:

Article Title: Tat Activates Human Immunodeficiency Virus Type 1 Transcriptional Elongation Independent of TFIIH Kinase

doi:

Figure Lengend Snippet: Strong and specific binding of Tat to P-TEFb but not to TFIIH. (A) Binding of P-TEFb to Tat. Affinity-purified P-TEFb complex fraction (6 μl) was incubated with wild-type GST-Tat(1-48) and three GST-Tat(1-48) mutants (C22G, K41A, and H33A) bound to glutathione-Sepharose beads. After extensive washes, the bound proteins were analyzed by SDS-PAGE and Western blotting for the presence of cyclin T1 and Cdk9-HA with antibodies specific for cyclin T1 and HA (MAb 12CA5), respectively. One microliter of the input P-TEFb fraction was used in lane 1 as a reference. The lower panel is a Coomassie-stained SDS gel showing the relative amounts of wild-type and mutant GST-Tat(1-48) proteins bound to glutathione-Sepharose beads. (B) Binding of TFIIH to Tat. TFIIH fraction (2 μl) with associated Cdk7-HA at a level similar to that of Cdk9-HA in P-TEFb (6 μl) was tested for binding to Tat under the same conditions as for P-TEFb. After washes, Western blotting with 12CA5 was used to detect Cdk7-HA bound to the GST-Tat beads. (C) Five times more TFIIH fraction (10 μl) was tested for binding to GST, wild-type, or mutant GST-Tat(1-48) beads. Bound proteins were examined by Western blotting with antibodies specific for ERCC3, the HA tag of Cdk7-HA, and Mat1.

Article Snippet: Commercial preparations of rabbit polyclonal antibodies directed against Cdk7, Mat1, and ERCC3 were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Binding Assay, Affinity Purification, Incubation, SDS Page, Western Blot, Staining, SDS-Gel, Mutagenesis

Journal:

Article Title: Tat Activates Human Immunodeficiency Virus Type 1 Transcriptional Elongation Independent of TFIIH Kinase

doi:

Figure Lengend Snippet: Efficient removal of CAK from holo-TFIIH by anti-Cdk7 depletion in the presence of high salt concentrations. (A) CAK can be dissociated from holo-TFIIH by high salt concentrations. Human 293T cells transiently transfected with an HA-tagged Cdk7 (Cdk7-HA) construct were lysed with buffers containing either 500 mM or 150 mM NaCl. Immunoprecipitation with anti-HA tag MAb 12CA5 was carried out at the same salt concentrations as in the lysis buffers. The immunoprecipitated proteins were analyzed by Western blotting with antibodies directed against Cdk7, Mat1, cyclin H, and ERCC3 as indicated. Occasionally, Cdk7-HA can be seen as a doublet probably because it can be modified differently under different conditions. (B) Cdk7-CAK in highly purified TFIIH complex can be immunodepleted in the presence of high salt concentrations. Highly purified TFIIH preparation was incubated with immobilized Cdk7 antibodies in the presence of 0.8 M KCl. Western blotting with antibodies directed against Cdk7 and ERCC3 was carried out to examine the presence of these two proteins in the highly purified TFIIH preparation after immunodepletion.

Article Snippet: Commercial preparations of rabbit polyclonal antibodies directed against Cdk7, Mat1, and ERCC3 were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Transfection, Construct, Immunoprecipitation, Lysis, Western Blot, Modification, Purification, Incubation

Journal:

Article Title: Tat Activates Human Immunodeficiency Virus Type 1 Transcriptional Elongation Independent of TFIIH Kinase

doi:

Figure Lengend Snippet: Tat transactivation in HeLa nuclear extract depleted of Cdk7 or Cdk9. (A [left panel]) Transcription reactions containing both templates pHIV+TAR-G400 and pHIVΔTAR-G100 were performed in the absence (−) or presence (+) of Tat and mock-depleted HeLa nuclear extract (lanes 1 and 2) or HeLa nuclear extract immunodepleted of the Cdk9 subunit of P-TEFb (lanes 3 to 6). Affinity-purified P-TEFb complex was added to Cdk9-depleted reactions as indicated. Immunodepletion was performed in the presence of 0.8 M KCl. (Right panel) The depleted extracts were examined by Western blotting with Cdk9 antibodies. (B and C) Cdk7, a subunit of the CAK ternary complex, was removed from HeLa nuclear extract by immunodepletion with anti-Cdk7 antibodies under high-salt (0.8 M KCl [B]) or low-salt (0.1 M KCl [C]) conditions. Immobilized anti-Myc antibody was used in control depletion reactions. (Left halves of panels B and C) Transcription reactions containing depleted extracts and transcription templates were carried out in the absence (−) or presence (+) of Tat. (Right halves of panels B and C) The depleted extracts were analyzed by Western blotting with anti-Cdk7 antibodies.

Article Snippet: Commercial preparations of rabbit polyclonal antibodies directed against Cdk7, Mat1, and ERCC3 were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Affinity Purification, Western Blot

Journal:

Article Title: Tat Activates Human Immunodeficiency Virus Type 1 Transcriptional Elongation Independent of TFIIH Kinase

doi:

Figure Lengend Snippet: Cdk7-containing complexes have different sizes at different salt concentrations. HeLa nuclear extracts were subjected to ultracentrifugation sedimentation through a 13 to 30% glycerol gradient containing either 0.1 M (B and D) or 0.8 M KCl (A and C). Gradient fractions were analyzed by Western blotting using antibodies specific for Cdk7 (A and B) or ERCC3 (C and D). A mixture of molecular mass marker proteins were sedimented in a parallel gradient. Their positions in the gradient were determined by SDS-PAGE and silver staining and are indicated in between panels B and C.

Article Snippet: Commercial preparations of rabbit polyclonal antibodies directed against Cdk7, Mat1, and ERCC3 were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Sedimentation, Western Blot, Marker, SDS Page, Silver Staining

Journal:

Article Title: Tat Activates Human Immunodeficiency Virus Type 1 Transcriptional Elongation Independent of TFIIH Kinase

doi:

Figure Lengend Snippet: Immunodepletion of CAK from holo-TFIIH with anti-Mat1 antibodies does not affect Tat activation. Immobilized anti-Mat1 antibodies were used to immunodeplete Mat1 from HeLa nuclear extract containing 0.8 M KCl. Immobilized anti-Myc MAb was used in a control depletion reaction. (A) The depleted extracts were analyzed for their abilities to mediate Tat activation in transcription reactions as described in the legend for Fig. ​Fig.1.1. (B) The removal of Mat1 and its associated Cdk7 in the CAK complex was confirmed by Western blotting with anti-Mat1 and anti-Cdk7 antibodies.

Article Snippet: Commercial preparations of rabbit polyclonal antibodies directed against Cdk7, Mat1, and ERCC3 were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Activation Assay, Western Blot