Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Utility of hemoglobin A1c in detecting risk of type 2 diabetes: comparison of hemoglobin A1c with other biomarkers

doi: 10.3164/jcbn.19-16

Figure Lengend Snippet: Correlation between the glucose and inslurin resistance during fasting and OGTT and fasting levels of multi-biomaekers

Article Snippet: HbA1c, glucose, insulin, leptin, adiponectin, RBP4, glycoalbumin, and hs-CRP were measured using commercially available ELISA kits {HbA1c: RAPIDIA Auto HbA1c-L [Fujirebio Inc. (Tokyo, Japan)]; glucose: Cica liquid GLU J [KANTO CHEMICAL Co., Inc. (Tokyo, Japan)]; insulin: Lumipulse Presto Insulin (Fujirebio Inc.); leptin: Human leptin RIA kit [Millipore Co., Inc. (Tokyo, Japan)]; adiponectin: CircuLexTM Human adiponectin ELISA Kit CY-8050 [MBL Co., Ltd. (Nagano, Japan)]; RBP4: CircuLex Human RBP4 ELISA Kit (MBL Co., Ltd.); glycoalbumin: Lucia GA-L [Asahi KASEI Pharma Co., Inc. (Tokyo, Japan)]; and hs-CRP: CircuLexTM Human HS-CRP ELISA Kit CY-8071 (MBL Co., Ltd.)}.

Techniques:

Journal:

Article Title: Heregulin Induces Transcriptional Activation of the Progesterone Receptor by a Mechanism That Requires Functional ErbB-2 and Mitogen-Activated Protein Kinase Activation in Breast Cancer Cells

doi: 10.1128/MCB.23.3.1095-1111.2003

Figure Lengend Snippet: Proliferative effects of HRG require PR and p42/p44 MAPK activation. C4HD (A) and T47D (B) cells were incubated for 48 h in medium with ChFCS supplemented, as indicated, with RU486, 10 nM MPA, MPA-RU486, HRG, HRG-RU486, HRG-ASODN or -SODN plus ErbB-2, or HRG-PD98059. C4HD cells were also treated with HRG plus increasing concentrations (1, 5, and 10 μg/ml) of either ErbB-3 monoclonal antibody (Ab-5) or ErbB-4 monoclonal antibody (Ab-3) and HRG plus preimmune mouse serum (NMS). Incorporation of [3H]thymidine was used as a measure of DNA synthesis. Data are presented as means ± the standard deviation (SD). Significance for columns b versus a, columns c versus b, columns d versus a, and columns e versus d, P < 0.001. The experiments shown are representative of a total of five for each cell type.

Article Snippet: Membranes were blocked with PBS-0.1% Tween 20 and immunoblotted with the following antibodies: ErbB-2 rabbit polyclonal antibody Neu C-18 (Santa Cruz Biotechnology, Santa Cruz, Calif.), ErbB-3 rabbit polyclonal antibody C-17 (Santa Cruz), ErbB-4 rabbit polyclonal antibody C-18 (Santa Cruz), ErbB-4/HER-4 oncoprotein Ab-2 rabbit polyclonal antibody (Neomarkers), anti-total p42/p44 MAPK (C-14; Santa Cruz) , anti-phospho p42/p44 MAPK (E-4 Santa Cruz), and antihemagglutinin (anti-HA) antibody HA.11 (Berkeley Antibody Co., Berkeley, Calif.).

Techniques: Activation Assay, Incubation, DNA Synthesis, Standard Deviation

Journal:

Article Title: Heregulin Induces Transcriptional Activation of the Progesterone Receptor by a Mechanism That Requires Functional ErbB-2 and Mitogen-Activated Protein Kinase Activation in Breast Cancer Cells

doi: 10.1128/MCB.23.3.1095-1111.2003

Figure Lengend Snippet: Blockage of ErbB-2 expression, ErbB-3 activation, or MAPK activity abolishes HRG capacity to induce PR binding to a PRE. (A) C4HD and T47D cells were treated for 2 h at 37°C with 10 nM MPA or HRG at 20 ng/ml or remained untreated and growing in ChFCS. ErbB-2 expression was blocked by pretreatment of both cell types for 48 h with 2 μM ASODN to ErbB-2 before the 2-h treatment with HRG. As a control, cells were preincubated with 2 μM SODN. MEK1/MAPK activity was abolished by preincubating the cells for 30 min with 10 μM PD98059 before treatment with HRG for 2 h. C4HD cells were also pretreated with 10 μg of ErbB-3 monoclonal antibody (Ab-5), ErbB-4 monoclonal antibody (Ab-3), or preimmune mouse serum (NMS)/ml before treatment with HRG for 2 h. Aliquots of nuclear extracts (4 fmol of PR) were incubated with a [32P]PRE oligonucleotide and subjected to an EMSA. (B) C4HD and T47D cells were incubated for 2 h with HRG at 20 ng/ml or preincubated for 48 h with 2 μM ErbB-2 ASODN or SODN and then treated for 2 h with HRG as described above. A total of 80 μg of protein from cell lysates was electrophoresed and immunoblotted for ErbB-2. Densitometric analysis of an ErbB-2 band expressed as percentages of the control value, i.e., cells growing in 20 ng of HRG/ml, yielded 32% for C4HD cells and 26% for T47D cells treated with 2 μM ASODN. No significant differences were found in the densitometric values of ErbB-2 bands between control cells and cells treated with 2 μM SODN. Autoradiograms from representative experiments of a total of three performed for each cell type are shown. (C to F) To evaluate the role of blocking antibodies on HRG-induced ErbB-3 and ErbB-4 tyrosine phosphoryation, C4HD cells were preincubated with 10 μg of either a ErbB-3 mouse monoclonal antibody (Ab-5) or a ErbB-4 mouse monoclonal antibody (Ab-3)/ml prior to HRG treatment for 10 min. As a control, cells were also incubated with preimmune normal mouse serum (NMS). ErbB-3 and ErbB-4 were immunoprecipitated as described in Materials and Methods, and immunocomplexes were subjected to SDS-PAGE (7.5% gel) and analyzed by Western blotting with an anti-P-Tyr monoclonal antibody (C and E). Identical aliquots of each immunoprecipitate were subjected to immunoblot analysis with anti-ErbB-3 (D) or ErbB-4 (F) antibodies to verify that nearly equal amounts of immunoprecipitated proteins were loaded. W, Western blot; IP, immunoprecipitation. (G) C4HD and T47D cells were incubated for 10 min with HRG at 20 ng/ml or were left untreated and growing in ChFCS. Blockage of MAPK activity was accomplished by preincubating the cells for 30 min with 10 μM PD98059 before stimulation with HRG. Cell lysates were immunoprecipitated by using an anti-p42/p44 MAPK antibody. The immunoprecipitates were assayed for MAPK activity with MBP as the substrate. (H) As loading control, identical aliquots of each immunoprecipitate assayed in panel G were subjected to immunoblot analysis with the anti-p42/p44 MAPK antibody. (I) C4HD and T47D cells were treated as described in panel G and, in addition, C4HD were treated with MPA for 10 min or were preincubated for 30 min with 10 μM PD98059 before stimulation with MPA. A total of 100 μg of protein from cell lysates was electrophoresed on 12% SDS-gels and immunoblotted with an anti-phospho p42/p44 MAPK antibody. (J) The membrane shown in panel I was stripped and hybridized with an antibody anti-total p42/p44 MAPK antibody.

Article Snippet: Membranes were blocked with PBS-0.1% Tween 20 and immunoblotted with the following antibodies: ErbB-2 rabbit polyclonal antibody Neu C-18 (Santa Cruz Biotechnology, Santa Cruz, Calif.), ErbB-3 rabbit polyclonal antibody C-17 (Santa Cruz), ErbB-4 rabbit polyclonal antibody C-18 (Santa Cruz), ErbB-4/HER-4 oncoprotein Ab-2 rabbit polyclonal antibody (Neomarkers), anti-total p42/p44 MAPK (C-14; Santa Cruz) , anti-phospho p42/p44 MAPK (E-4 Santa Cruz), and antihemagglutinin (anti-HA) antibody HA.11 (Berkeley Antibody Co., Berkeley, Calif.).

Techniques: Expressing, Activation Assay, Activity Assay, Binding Assay, Incubation, Blocking Assay, Immunoprecipitation, SDS Page, Western Blot

Journal:

Article Title: Heregulin Induces Transcriptional Activation of the Progesterone Receptor by a Mechanism That Requires Functional ErbB-2 and Mitogen-Activated Protein Kinase Activation in Breast Cancer Cells

doi: 10.1128/MCB.23.3.1095-1111.2003

Figure Lengend Snippet: HRG induces transcriptional activity of PR. C4HD (A) and T47D (B) cells were transiently transfected with 4 μg of a PRE2-tk-CAT reporter plasmid/well and with 4 μg of a CMV-β-Gal expression vector/well as an internal control. C4HD cells were also transfected with a tk-CAT reporter lacking a PRE. Cells were then treated with MPA, MPA-RU486, HRG, and HRG-RU486 at 37°C for 48 h for C4HD cells or for 24 h for T47D cells or were left untreated and growing in ChFCS. To study the effect of ErbB-2 expression blockage, 2 μM ErbB-2 ASODN or 2 μM SODN was added, along with HRG, during the 48-h treatment. To inhibit MAPK activation, cells were preincubated with 10 μM PD98059 before HRG treatment. C4HD cells were also pretreated with 10 μg of ErbB-3 monoclonal antibody (Ab-5) or ErbB-4 monoclonal antibody (Ab-3)/ml or preimmune mouse serum before treatment with HRG. Cells were harvested and lysed, and the CAT and β-galactosidase activities were measured as described in Materials and Methods. The results are presented as the percent activation, where 100% represents the maximal activity of PR in the presence of MPA. The data shown represent the means of six independent experiments ± the SEM. Significance for columns b versus a, columns c versus b, columns d versus a, and columns e versus d, P < 0.001.

Article Snippet: Membranes were blocked with PBS-0.1% Tween 20 and immunoblotted with the following antibodies: ErbB-2 rabbit polyclonal antibody Neu C-18 (Santa Cruz Biotechnology, Santa Cruz, Calif.), ErbB-3 rabbit polyclonal antibody C-17 (Santa Cruz), ErbB-4 rabbit polyclonal antibody C-18 (Santa Cruz), ErbB-4/HER-4 oncoprotein Ab-2 rabbit polyclonal antibody (Neomarkers), anti-total p42/p44 MAPK (C-14; Santa Cruz) , anti-phospho p42/p44 MAPK (E-4 Santa Cruz), and antihemagglutinin (anti-HA) antibody HA.11 (Berkeley Antibody Co., Berkeley, Calif.).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Activation Assay