Journal: Biomedicines

Article Title: MicroRNA Associated with the Invasive Phenotype in Clear Cell Renal Cell Carcinoma: Let-7c-5p Inhibits Proliferation, Migration, and Invasion by Targeting Insulin-like Growth Factor 1 Receptor

doi: 10.3390/biomedicines10102425

Figure Lengend Snippet: The effect of let-7c-5p on the expression of IGF1R. ( A ) Relative luciferase activity of Caki-1 cells for each vector, including mutants, transfected with pre-let-7c-5p, relative to the negative control transfectant (predicted sites detailed in the inset below the chart). ( B ) Relative expression of IGF1R in Caki-1 and 786-O cells transfected with let-7c-5p after 48 h, relative to the negative control for each cell line. Representative image inset to the right: the first lane of each pair corresponds to IGF1R and the second is total protein detected for the same lane. * p < 0.05, ** p < 0.01, and NS = not significant.

Article Snippet: IGF1R primary antibody (NBP1-77679, Novus Biologicals, Littleton, CO, USA) at a concentration of 1:25 diluted in Antibody diluent 2 (ProteinSimple) and rabbit secondary antibody (042-206, ProteinSimple, used as provided) were added according to the manufacturer’s protocol.

Techniques: Expressing, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Negative Control

Journal:

Article Title: Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

doi: 10.1128/MCB.22.3.965-977.2002

Figure Lengend Snippet: Effect of disruption of Pik3r1 on the interaction between the regulatory subunit and phosphorylated IRS proteins in response to IGF-1. (a) Protein and phosphorylation levels of the IGF-1 receptor in cells of each genotype. The cells were starved for 24 h and then stimulated with 10 nM IGF-1 for 5 min. Cell lysates were subjected to immunoprecipitation with αIGF-1R followed by immunoblotting with αIGF-1R (top panel) or 4G10 (bottom panel). (b) Interaction between IRS proteins and the regulatory subunit. Cell lysates were subjected to immunoprecipitation with anti-IRS-1 (αIRS-1; left panels), anti-IRS-2 (αIRS-2; middle panels), or anti-Gab-1 (αGab-1; right panels) antibody followed by immunoblotting with the same antibody (top panels), 4G10 antibody (middle panels), or αp85pan antibody (bottom panels). Wild, wild type; hetero, heterozygous KO.

Article Snippet: Rabbit polyclonal anti-p85α (αp85pan) antibodies and mouse monoclonal anti-p85α (αp85α) antibodies were purchased from Upstate Biotechnology, Inc. Rabbit polyclonal anti-IRS-1 antibodies and anti-IRS-2 antibodies were generated as described previously ( 6 ), and rabbit polyclonal anti-IGF-1 receptor (αIGF-1R) antibodies and anti-Gab-1 antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot

Journal:

Article Title: Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

doi: 10.1128/MCB.22.3.965-977.2002

Figure Lengend Snippet: Effect of disruption of Pik3r1 on the PI 3-kinase activity associated with each signaling molecule. (a) PI 3-kinase activities associated with the regulatory subunits. The cells were starved for 24 h and then stimulated with 10 nM IGF-1 for 5 min. Cell lysates were subjected to immunoprecipitation with αp85pan (left panels), αp85α (middle panels), or αp85β (right panels) antibody followed by the PI 3-kinase assay. The top panels show representative results, and in the bottom panels each bar represents the mean ± standard deviation of the relative PI-3 kinase activity calculated from the results of three independent experiments. In the αp85pan precipitation: *, P value of <0.05 for wild-type (Wild) versus null cells. In the p85β precipitation: *, P value of <0.01 for wild versus heterozygous KO (Hetero) cells; **, P value of <0.01 for wild versus null cells. (b) PI 3-kinase activities associated with the catalytic subunit and tyrosine-phosphorylated proteins. Cell lysates were subjected to immunoprecipitation with αp110α (left panels) or 4G10 (right panels) antibody followed by the PI 3-kinase assay. Top panels show representative results, and in the bottom panels each bar represents the mean ± standard deviation of the relative PI-3 kinase activity calculated from the results of three independent experiments. *, P value of <0.01 for wild versus null cells.

Article Snippet: Rabbit polyclonal anti-p85α (αp85pan) antibodies and mouse monoclonal anti-p85α (αp85α) antibodies were purchased from Upstate Biotechnology, Inc. Rabbit polyclonal anti-IRS-1 antibodies and anti-IRS-2 antibodies were generated as described previously ( 6 ), and rabbit polyclonal anti-IGF-1 receptor (αIGF-1R) antibodies and anti-Gab-1 antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Immunoprecipitation, Kinase Assay, Standard Deviation

Journal:

Article Title: Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

doi: 10.1128/MCB.22.3.965-977.2002

Figure Lengend Snippet: Effect of disruption of Pik3r1 on production of PIP3 in response to IGF-1 in vivo. (a) IGF-1-induced PIP3 production in cells of each genotype. Cells were labeled with [32P]orthophosphate as described in Materials and Methods and stimulated with 10 nM IGF-1 for the indicated period. 32P-labeled phospholipids were extracted and separated by TLC. In the graph, the mean levels of PIP3 normalized to the total labeled phospholipids from two independent experiments are shown. (b) Time course of PI 3-kinase associated with phosphotyrosine complex in cells of each genotype. Cells were stimulated with 10 nM IGF-1 for the indicated period and subjected to immunoprecipitation with 4G10 followed by the PI 3-kinase assay. (c) Expression levels of PTEN and SHIP in cells of each genotype. Cell lysates were subjected to immunoblotting with anti-PTEN (αPTEN; top panel) or anti-SHIP (αSHIP; bottom panel) antibody. Wild, wild type; Hetero, heterozygous KO.

Article Snippet: Rabbit polyclonal anti-p85α (αp85pan) antibodies and mouse monoclonal anti-p85α (αp85α) antibodies were purchased from Upstate Biotechnology, Inc. Rabbit polyclonal anti-IRS-1 antibodies and anti-IRS-2 antibodies were generated as described previously ( 6 ), and rabbit polyclonal anti-IGF-1 receptor (αIGF-1R) antibodies and anti-Gab-1 antibodies were purchased from Santa Cruz Biotechnology.

Techniques: In Vivo, Labeling, Immunoprecipitation, Kinase Assay, Expressing, Western Blot

Journal:

Article Title: Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

doi: 10.1128/MCB.22.3.965-977.2002

Figure Lengend Snippet: Effect of disruption of Pik3r1 on downstream kinases from PI 3-kinase. (a) IGF-1-induced Akt activity in cells of each genotype. Cells were starved for 24 h and then stimulated with 10 nM IGF-1 for 5 min. Cell lysates were subjected to immunoblotting with anti-phospho-Akt (αphospho-Akt; top panel) antibody or immunoprecipitation with αAkt antibody. The immunoprecipitates were subjected to an immune complex kinase assay. In the bottom panel, each bar represents the mean ± standard deviation of the relative Akt kinase activity calculated from the results of three independent experiments. *, P value of <0.01 for wild-type (Wild) versus heterozygous KO (Hetero) cells; **, P value of <0.05 for wild versus null cells. (b) IGF-1-induced p70S6K activity in cells of each genotype. After 20 min of stimulation with 10 nM IGF-1, cell lysates were subjected to immunoblotting with anti-phospho-p70S6K (αphospho-p70S6K; top panel) antibody or immunoprecipitation with αp70S6K antibody. The immunoprecipitates were subjected to an immune complex kinase assay. In the bottom panel, each bar represents the mean ± standard deviation of the relative p70S6K kinase activity calculated from the results of three independent experiments.

Article Snippet: Rabbit polyclonal anti-p85α (αp85pan) antibodies and mouse monoclonal anti-p85α (αp85α) antibodies were purchased from Upstate Biotechnology, Inc. Rabbit polyclonal anti-IRS-1 antibodies and anti-IRS-2 antibodies were generated as described previously ( 6 ), and rabbit polyclonal anti-IGF-1 receptor (αIGF-1R) antibodies and anti-Gab-1 antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Immune Complex Kinase Assay, Standard Deviation

Journal:

Article Title: Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

doi: 10.1128/MCB.22.3.965-977.2002

Figure Lengend Snippet: Effect of disruption of Pik3r1 on serum deprivation-induced apoptosis and IGF-1-dependent antiapoptosis. Cells were cultured in the indicated concentration of serum (left panel) or IGF-1 without serum (right panel) for 5 h. The level of apoptosis was assessed using an enzyme-linked immunosorbent assay for nucleosomal DNA as described in Materials and Methods. Each value is expressed as the ratio of the value of the wild-type cells treated with 10% serum and represents the mean ± standard deviation of three independent experiments. Wild, wild type; Hetero, heterozygous KO.

Article Snippet: Rabbit polyclonal anti-p85α (αp85pan) antibodies and mouse monoclonal anti-p85α (αp85α) antibodies were purchased from Upstate Biotechnology, Inc. Rabbit polyclonal anti-IRS-1 antibodies and anti-IRS-2 antibodies were generated as described previously ( 6 ), and rabbit polyclonal anti-IGF-1 receptor (αIGF-1R) antibodies and anti-Gab-1 antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal:

Article Title: Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

doi: 10.1128/MCB.22.3.965-977.2002

Figure Lengend Snippet: Effect of disruption of Pik3r1 on IGF-1-dependent antiapoptotic signaling. (a) IGF-1-induced Bad phosphorylation and the interaction between Bad and 14-3-3 in cells of each genotype. Cells were starved for 24 h and then stimulated with 10 nM IGF-1 for 20 min. Cell lysates were subjected to immunoprecipitation with αBad antibody followed by immunoblotting. Immunoblots were probed with αBad (top panel), anti-phospho-Bad (αphospho-Bad; middle panel), or anti-14-3-3 (α14-3-3; bottom panel) antibody and visualized by enhanced chemiluminescence with protein A-conjugated peroxidase. (b) IGF-1-induced p90RSK activity in cells of each genotype. After 20 min of stimulation with 10 nM IGF-1, cell lysates were subjected to immunoprecipitation with αp90RSK antibody followed by an immune complex kinase assay. Each bar represents the mean ± standard deviation of the p90RSK activity calculated from the results of three independent experiments. *, P value of <0.01 for wild-type (Wild) versus null cells. (c) IGF-1-induced FKHR phosphorylation. After 20 min of stimulation with 10 nM IGF-1, cell lysates were subjected to immunoblotting with anti-phospho-FKHR (αphospho-FKHR; top panel) or anti-FKHR (αFKHR; bottom panel) antibody. (d) IGF-1-induced CREB phosphorylation in cells of each genotype. After 20 min of stimulation with 10 nM IGF-1, cell lysates were subjected to immunoblotting with anti-phospho-CREB (αphospho-CREB; top panel) or anti-CREB (αCREB; bottom panel) antibody. Hetero, heterozygous KO.

Article Snippet: Rabbit polyclonal anti-p85α (αp85pan) antibodies and mouse monoclonal anti-p85α (αp85α) antibodies were purchased from Upstate Biotechnology, Inc. Rabbit polyclonal anti-IRS-1 antibodies and anti-IRS-2 antibodies were generated as described previously ( 6 ), and rabbit polyclonal anti-IGF-1 receptor (αIGF-1R) antibodies and anti-Gab-1 antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot, Activity Assay, Immune Complex Kinase Assay, Standard Deviation