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Vector Labs  
Product Name
Saccharomyces cerevisiae Meyen ex E C Hansen
Catalog Number
4023352
Biosafety Level
1
Genotype
MATa/MATalpha his3delta1/his3delta1 leu2delta0/leu2delta0 lys2delta0/+ met15delta0/+ ura3delta0/ura3delta0 deltaNGR1
Category
Fungi
Product Format
Frozen
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Activation Assay
Activity Assay
Expressing
Immunoprecipitation
In Vitro
Infection
Over Expression
Plasmid Preparation
Western Blot
Epidermal Growth Factor Receptor-Dependent Regulation of Integrin-Mediated Signaling and Cell Cycle Entry in Epithelial Cells 
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FN-induced EGFR activation is required for G1 cell cycle entry. (A and B) Adherent CV1 cells were serum starved for more than 48 h (Go), placed in suspension (S), left untreated or treated with 1 μM AG1478 (AG), and plated on FN in the absence (FN) or presence (EGF) of 40 ng of EGF/ml for 12 or 18 h. The time points selected were determined to be optimal for the events monitored in time course studies (data not shown). (A) The levels of <t>cyclin</t> <t>D1</t> (cycD1), cyclin E (cycE), p27, p21, and Rb phosphorylation at Ser807/811 (P-Rb) at 12 h and the levels of cyclin A at 18 h were monitored by immunoblotting cell lysates with their respective antibodies. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts. (B) cdk2 activity was measured by using in vitro kinase assays with histone H1 as a substrate and was quantified (1.74- ± 0.03-fold induction in the presence of FN only; 2.69- ± 0.01-fold in the presence of FN and EGF; n = 3). Preimmune (PI) and no-substrate (NS) controls were included. Total levels of kinase immunoprecipitated in the assays were monitored by immunoblotting with anti-cdk2 antibodies (cdk2). (C and D) CV1 cells were prepared as described above except that cells in suspension were pretreated with either 10 μM U0126 (U0), 100 nM wortmannin (WT), or 0.5 μM PD168393 (PD) 15 min prior to plating on FN. Results obtained after plating for 12 h are shown. (C) cdk4 kinase activity was monitored by an in vitro kinase assay by measuring phosphate incorporation into GST-Rb 12 h after plating on FN and was quantified (1.4- ± 0.2-fold in the presence or absence of EGF; n = 3). Preimmune and no-substrate controls were included. Total levels of kinase immunoprecipitated in the assays were monitored by immunoblotting with anti-cdk4 antibodies (cdk4). (D) Levels of cyclin D1, p27, and Rb phosphorylation at Ser807/811 were monitored by immunoblotting cell lysates with their respective antibodies. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts.
Epidermal Growth Factor Receptor-Dependent Regulation of Integrin-Mediated Signaling and Cell Cycle Entry in Epithelial Cells 
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EGFR and Myc overexpression rescues S phase. (A) CV1 cells were serum starved for 48 h and either left in suspension (S) or plated on FN in the absence (FN) or presence (EGF) of 40 ng of EGF/ml for the indicated times. The levels of <t>cyclin</t> <t>D1</t> (cycD1), p27, cyclin A (cycA), and Rb phosphorylation at Thr821 (P-Rb T821) were monitored by immunoblotting with the respective antibodies. Total levels of Akt were monitored to control for total protein levels. (B) CV1 cells (CV) were infected with retroviruses expressing EGFR (R1, R5) or cyclin D1 (D1) or with an empty vector (V). Levels of EGFR and cyclin D1 expression were monitored by immunoblotting of whole-cell extracts with their respective antibodies after plating of cells on FN for 12 h. Levels of EGFR activation in virus-infected cells held in suspension or attached to FN were monitored by immunoblotting of EGFR immunoprecipitates with anti-phosphotyrosine antibodies (P-Tyr Blot). Total levels of EGFR were monitored by immunoblotting with an anti-EGFR antibody (EGFR Blot). *, the length of film exposure to enhanced chemiluminescence was 5 times less for EGFR-overexpressing cells than for other samples. (C) Adherent CV1 cells infected with retroviral vectors encoding either EGFR, cyclin D1, or no cDNA (Vector) were serum starved for 48 h and either placed in suspension or plated on FN for the indicated times. Levels of cyclin A and p27 were monitored by immunoblotting cell lysates with their respective antibodies. Rb phosphorylation at Thr821 was monitored by immunoblotting of cell lysates with phosphospecific antibodies. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts. (D) CV1 cells were serum starved for 48 h and either placed in suspension or plated on FN in the absence or presence of 40 ng of EGF/ml for the indicated times. Adherent CV1 cells infected with retroviral vectors encoding EGFR or no cDNA were serum starved for 48 h and then either placed in suspension or plated on FN for the indicated times. Levels of Myc expression were monitored by immunoblotting of cell lysates with anti-Myc antibodies. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts. (E) Adherent CV1 cells infected with retroviral vectors encoding either EGFR or no cDNA were serum starved for 48 h and then either placed in suspension or plated on FN for the indicated times. Levels of cyclin D1 were monitored by immunoblotting of cell lysates with anti-cyclin D1 antibodies. Erk and Akt activation was monitored by immunoblotting of cell lysates with phosphospecific antibodies (P-Erk, P-Akt). Total levels of Erk and Akt were monitored by immunoblotting as a control for total protein levels in the extracts (Erk, Akt). (F) CV1 cells were infected with a virus containing an empty vector or expressing MycER. Levels of MycER expression were monitored by immunoblotting with anti-Myc antibodies after plating on FN in the absence (FN) or presence (Tx) of 50 nM tamoxifen. Levels of p27 and cyclin A expression in cells in suspension or at the indicated times after plating on FN in the presence or absence of 50 nM tamoxifen were monitored by immunoblotting. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts.
Epidermal Growth Factor Receptor-Dependent Regulation of Integrin-Mediated Signaling and Cell Cycle Entry in Epithelial Cells 
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Adhesion stimulates G1 cell cycle events in primary cells. Adherent primary prostate epithelial cells (A) or primary keratinocytes (B) were serum starved for 24 h, placed in suspension (S), left untreated or treated with 0.5 μM PD168393 (PD), and plated on FN in the absence (FN) or presence (EGF) of 40 ng of EGF/ml for 18 h. Levels of <t>cyclin</t> <t>D1</t> (cycD1), cyclin A (cycA), p27, p21, and Rb phosphorylation (P-Rb) were monitored by immunoblotting cell lysates with their respective antibodies. Note that the levels of Rb phosphorylation are less than optimal due to the late time point selected for this assay. *, cyclin D1 is the lower band. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts.
Epidermal Growth Factor Receptor-Dependent Regulation of Integrin-Mediated Signaling and Cell Cycle Entry in Epithelial Cells , 2004 Oct 01
"FN-induced EGFR activation is required for G1 cell cycle entry. (A and B) Adherent CV1 cells were serum starved for more than 48 h (Go), placed in suspension (S), left untreated or treated with 1 μM AG1478 (AG), and plated on FN in the absence (FN) or presence (EGF) of 40 ng of EGF/ml for 12 or 18 h. The time points selected were determined to be optimal for the events monitored in time course studies (data not shown). (A) The levels of <t>cyclin</t> <t>D1</t> (cycD1), cyclin E (cycE), p27, p21, and Rb phosphorylation at Ser807/811 (P-Rb) at 12 h and the levels of cyclin A at 18 h were monitored by immunoblotting cell lysates with their respective antibodies. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts. (B) cdk2 activity was measured by using in vitro kinase assays with histone H1 as a substrate and was quantified (1.74- ± 0.03-fold induction in the presence of FN only; 2.69- ± 0.01-fold in the presence of FN and EGF; n = 3). Preimmune (PI) and no-substrate (NS) controls were included. Total levels of kinase immunoprecipitated in the assays were monitored by immunoblotting with anti-cdk2 antibodies (cdk2). (C and D) CV1 cells were prepared as described above except that cells in suspension were pretreated with either 10 μM U0126 (U0), 100 nM wortmannin (WT), or 0.5 μM PD168393 (PD) 15 min prior to plating on FN. Results obtained after plating for 12 h are shown. (C) cdk4 kinase activity was monitored by an in vitro kinase assay by measuring phosphate incorporation into GST-Rb 12 h after plating on FN and was quantified (1.4- ± 0.2-fold in the presence or absence of EGF; n = 3). Preimmune and no-substrate controls were included. Total levels of kinase immunoprecipitated in the assays were monitored by immunoblotting with anti-cdk4 antibodies (cdk4). (D) Levels of cyclin D1, p27, and Rb phosphorylation at Ser807/811 were monitored by immunoblotting cell lysates with their respective antibodies. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts. "
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