Journal:

Article Title: Cell-Extracellular Matrix Interactions Stimulate the AP-1 Transcription Factor in an Integrin-Linked Kinase- and Glycogen Synthase Kinase 3-Dependent Manner

doi:

Figure Lengend Snippet: ILK-induced AP-1 activity is mediated by c-jun. (A) In this band shift assay, serum-exposed HEK-293 cells were transfected by Lipofectin with 0.5 μg of each of the indicated cDNAs. PD98059 (25 μM) was added to the medium 12 h prior to harvesting the cells. Nuclear extracts (2 μg) from the transfected cells were incubated with 32P-end-labeled AP-1 consensus oligonucleotide containing the protein binding site. Reaction products were analyzed on a nondenaturing 5% polyacrylamide gel (left gel). The specificity of complex formation was established by a competition experiment using cold AP-1 oligonucleotide as the competitor (right gel). For immunoblot studies, 10 μg of protein was resolved by SDS–10% PAGE. c-jun (Ser-73) phosphorylation, c-jun protein expression level, ERK phosphorylation (Tyr-204), and ERK protein expression level (not shown) were determined by Western blot analysis. ILK and GSK-3 expression levels in the transfected cells were evaluated by Western blot analysis using anti-V5 and anti-HA antibodies, respectively. (B) Anti-c-jun antibody shifts ILK-induced AP-1 complex. Nuclear extracts (2 μg) from HEK-293 cells transfected with ILK-V5 cDNA were incubated with 32P-AP-1 oligonucleotide in the absence or presence of anti-c-jun antibody or nonspecific IgG (10 μg). The latter did not induce a mobility shift of the complex (not shown).

Article Snippet: For the supershift assay, 10 μg of rabbit anti-c-jun antibody (Santa Cruz Biotechnology) or nonspecific IgG was added to the reaction mixture, subsequent to the addition of the 32 P-labeled oligonucleotide probe, and the mixture was incubated for 45 min at room temperature.

Techniques: Activity Assay, Electrophoretic Mobility Shift Assay, Transfection, Incubation, Labeling, Protein Binding, Western Blot, Expressing, Mobility Shift

Journal:

Article Title: Inhibition of T-Cell Acute Lymphoblastic Leukemia Proliferation In Vivo by Re-expression of the p16 INK4a Tumor Suppressor Gene

doi:

Figure Lengend Snippet: Expression of p16INK4a (A) and pRB (B) in T-ALL cell lines. (A) Lysates from murine T-ALL cell lines were immunoprecipitated by using a monoclonal antibody that recognizes mouse p16INK4a or PAb 419 as a nonspecific control. Samples were resolved by SDS-PAGE and subjected to immunoblot analysis using the same p16INK4a antibody. (B) Lysates from murine T-ALL cells were subjected to immunoblot analysis with the anti-Rb antibody G3-245 (Pharmingen). Lysates from Balb/c 3T3 cells were used as a positive control. Both the hypo- and hyperphosphorylated forms of pRB can be seen in each sample. Size markers are indicated on the left.

Article Snippet: One half of each lysate was incubated with the anti-simian virus 40 Large T antigen antibody, pAb 419 [ 24 ] as a nonspecific control, or with the monoclonal p16 INK4a antibody M-156 (2 μg per sample; Santa Cruz Biotechnology, Inc) overnight at 4°C.

Techniques: Expressing, Immunoprecipitation, Control, SDS Page, Western Blot, Positive Control

Journal:

Article Title: Inhibition of T-Cell Acute Lymphoblastic Leukemia Proliferation In Vivo by Re-expression of the p16 INK4a Tumor Suppressor Gene

doi:

Figure Lengend Snippet: Expression of hp16INK4a in MSCV-hp16INK4a-infected T-ALL cells (A) and binding of human p16INK4a to murine CDK4 (B). (A) Lysates from MSCV-hp16INK4a-infected and from uninfected T-ALL 4, T-ALL 5, and T-ALL 68 cells were immunoprecipitated with a polyclonal antibody that specifically recognizes human but not murine p16INK4a or with pAb 419 as a nonspecific control. Immunoprecipitates were resolved by SDS-PAGE and subjected to immunoblot analysis with the same p16INK4a antibody. Uninfected cells were used as negative controls and the human glioblastoma cell line D384 was used as a positive control for hp16INK4a. (B) Lysates from infected and uninfected T-ALL 68 cells were immunoprecipitated as in (A), and samples were subjected to immunoblot analysis with a monoclonal antibody that binds murine and human (weakly) CDK4. D384 was used as a control for binding of human CDK4 by human p16INK4a. Size markers are indicated on the left.

Article Snippet: One half of each lysate was incubated with the anti-simian virus 40 Large T antigen antibody, pAb 419 [ 24 ] as a nonspecific control, or with the monoclonal p16 INK4a antibody M-156 (2 μg per sample; Santa Cruz Biotechnology, Inc) overnight at 4°C.

Techniques: Expressing, Infection, Binding Assay, Immunoprecipitation, Control, SDS Page, Western Blot, Positive Control

Journal:

Article Title: Inhibition of T-Cell Acute Lymphoblastic Leukemia Proliferation In Vivo by Re-expression of the p16 INK4a Tumor Suppressor Gene

doi:

Figure Lengend Snippet: MSCV-hp16INK4a suppresses the proliferation of infected murine T-ALL cell lines. T-ALL 4 (A), T-ALL 5 (B), and T-ALL 68 (C) were infected as described. The rate of proliferation was determined by labeling with [3H]-thymidine for 6 hours on the indicated days. The amount of [3H]-thymidine incorporated for each infected cell line is plotted versus the number of days after infection. The status of endogenous p16INK4a is indicated for each line.

Article Snippet: One half of each lysate was incubated with the anti-simian virus 40 Large T antigen antibody, pAb 419 [ 24 ] as a nonspecific control, or with the monoclonal p16 INK4a antibody M-156 (2 μg per sample; Santa Cruz Biotechnology, Inc) overnight at 4°C.

Techniques: Infection, Labeling

Journal:

Article Title: Inhibition of T-Cell Acute Lymphoblastic Leukemia Proliferation In Vivo by Re-expression of the p16 INK4a Tumor Suppressor Gene

doi:

Figure Lengend Snippet: Expression of human p16INK4a in murine T-ALL cell lines induces G1-arrest. T-ALL 4 (A), T-ALL 5 (B), and T-ALL 68 (C) cells were infected as described. On days 1, 2, and 3 after infection, the fraction of cells in the G1- , S- , and G2-phases of the cell cycle was determined by BrdU incorporation and staining with PI. Those cells with a DNA content less than 2N as determined by PI staining were considered to be sub G1. The status of endogenous p16INK4a is indicated for each line.

Article Snippet: One half of each lysate was incubated with the anti-simian virus 40 Large T antigen antibody, pAb 419 [ 24 ] as a nonspecific control, or with the monoclonal p16 INK4a antibody M-156 (2 μg per sample; Santa Cruz Biotechnology, Inc) overnight at 4°C.

Techniques: Expressing, Infection, BrdU Incorporation Assay, Staining