rabbit monoclonal anti fabp4 ap2  (Cell Signaling Technology Inc)

Journal: PLoS ONE

Article Title: Endoribonuclease L (RNase L) Regulates the Myogenic and Adipogenic Potential of Myogenic Cells

doi: 10.1371/journal.pone.0007563

Figure Lengend Snippet: A: C2-RNase L cells were treated (+RNase L) or not (Control) with 5 mM IPTG for 6 h at day 0 (panels a and b), and then were induced to differentiate in MDM for 4 (panels c,d,i,j) or 6 days (panels e and f and panels g and h). At the different time points cells were fixed and incubated with a monoclonal antibody against Troponin-T (panels a to f) or stained with Oil-red-O (panels g and h). At day 4, cells were also analyzed with a monoclonal antibody against Fabp4/aP2 (panels i and j). Cells were observed at a magnification of 20x, ( __ ): 60 µm. B: At day 1, C2-RNase L cells were treated (+RNase L) or not (Control) with 5 mM IPTG for 6 h and then were induced to differentiate in MDM. Cells were then collected at the indicated time points and expression of specific mRNAs was analyzed by RT-PCR amplification. PCR products were analyzed on 1.2% agarose gels. Photographs of the gels are shown. RNase L protein expression was analyzed as in . Autoradiographs of these gels are shown (RNase L-P). C: Quantification of lipids accumulated in C2-RNase L cells treated ( ) or not ( ) with 5 mM IPTG for 6 h and then induced to differentiate in MDM for 6 days. After staining with Oil-red-O, lipids were quantified by spectrophotometric analysis at 540 mm following elution of cell-retained Oil-red-O with isopropanol. The amount of lipids in untreated C2-RNase L cells was set to 1. Data were expressed as mean±SD of three different experimental points. (*) P<0.01 compared to non-induced cells.

Article Snippet: After blocking with 10% (W/V) FCS in PBS, cells were incubated with a mouse monoclonal anti-Troponin T antibody (1/1000; Sigma) or a rabbit monoclonal anti-Fabp4/aP2 (1/100; Cell Signaling) at room temperature for 1 h. Cells were then washed and incubated with a donkey anti-mouse secondary antibody conjugated to TRITC or FITC (Santa-Cruz) or a donkey anti-rabbit secondary antibody conjugated to TRITC (Santa-Cruz) at room temperature for 1 h. For adipocyte differentiation, cells were cultured in adipocyte differentiating medium (ADM) [DMEM-10% (V/V) FCS, 5 µg/ml Bovine insulin (Sigma) and 1 µM dexamethazone (Sigma)] for 6 days.

Techniques: Incubation, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: PLoS ONE

Article Title: Endoribonuclease L (RNase L) Regulates the Myogenic and Adipogenic Potential of Myogenic Cells

doi: 10.1371/journal.pone.0007563

Figure Lengend Snippet: A: C2-RNase L cells were treated (+RNase L) or not (Control) with 5 mM IPTG for 6 h at day 0 and then induced to differentiate in ADM. Cells were fixed and stained with Oil-red-O to reveal lipids accumulation at day 0, 3 and 6. Cells were observed at a magnification of 20x, ( __ ): 60 µm. B: Quantification of lipids in control ( ) or IPTG-treated C2-RNase L cells ( ) at day 0, 3 and 6 after induction of differentiation in ADM. After staining with Oil-red-O, lipids were quantified by spectrophotometric analysis at 540 mm following elution of cell-retained Oil-red-O with isopropanol. The amount of lipids in C2-RNase L cells at T = 0, before addition of IPTG, was set to 1. Error bars refer to the standard deviation obtained in three independent experiments. (*) P<0.01 compared to untreated cells at T = 3. C: C2-RNase L cells were treated (+RNase L) or not (Control) with 5 mM IPTG for 6 h at day 0 and then induced to differentiate in ADM. Cells were fixed at day 0, 3 and 6 and then incubated with an antibody against Fabp4/aP2 (Red). DNA was stained with Dapi (Blue). Cells were observed at 20x, ( __ ): 50 µm. D: Analysis of mRNA expression by RT-PCR amplification at different time points during adipocyte differentiation. RNase L was induced (+RNase L) or not (Control) with 5 mM IPTG for 6 h at day 2 and then cells were switched to ADM to induce adipocyte differentiation. PCR products were analyzed by gel electrophoresis. Photographs of the gels are shown.

Article Snippet: After blocking with 10% (W/V) FCS in PBS, cells were incubated with a mouse monoclonal anti-Troponin T antibody (1/1000; Sigma) or a rabbit monoclonal anti-Fabp4/aP2 (1/100; Cell Signaling) at room temperature for 1 h. Cells were then washed and incubated with a donkey anti-mouse secondary antibody conjugated to TRITC or FITC (Santa-Cruz) or a donkey anti-rabbit secondary antibody conjugated to TRITC (Santa-Cruz) at room temperature for 1 h. For adipocyte differentiation, cells were cultured in adipocyte differentiating medium (ADM) [DMEM-10% (V/V) FCS, 5 µg/ml Bovine insulin (Sigma) and 1 µM dexamethazone (Sigma)] for 6 days.

Techniques: Staining, Standard Deviation, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Nucleic Acid Electrophoresis

Journal: PLoS ONE

Article Title: Endoribonuclease L (RNase L) Regulates the Myogenic and Adipogenic Potential of Myogenic Cells

doi: 10.1371/journal.pone.0007563

Figure Lengend Snippet: A: C2-RNase L cells were treated, or not (0), with 5 mM IPTG for (2) or (6)h. Cells were then harvested and analyzed for RNase L 2-5A binding activity with the 2-5A radio-covalent binding assay. Proteins were separated on 10% polyacrylamide gels. A representative autoradiography of the gel and a densitometry of the gel are shown. The amount of 2-5A binding activity in C2-RNase L cells in the absence of IPTG was set to 1. Error bars refer to the standard deviation obtained in three independent experiments. (*) P<0.0 compared to non- induced cells (**) P<0.01 compared to control, untreated cells. B: RNase L was induced by 5 mM IPTG for 0, 2 or 6 hours, and then mRNA expression was analyzed by RT-PCR amplification and agarose gel electrophoresis. Photographs and densitometric analysis of the gels are shown. The level of the different mRNAs in untreated cells was set at 1. Quantification of EEF1α mRNA was used as a control of semi-quantitative RT-PCR experiment. Error bars refer to the standard deviation obtained in three independent experiments. C: C2-RNase L cells were treated, or not, with 5 mM IPTG for 2 or 6 h and then induced to differentiate in ADM. At T = 0, T = 2, T = 4 and T = 6 days after induction of differentiation, cells were fixed and stained with Oil-red-O to reveal lipid accumulation (panel on the right) or incubated with an antibody against Fabp4/aP2 (Red) (panel on the left); DNA was stained with Dapi (Blue). Cells were observed at 20x, ( __ ): 50 µm. D: Quantification of lipids in control or IPTG-treated C2-RNase L cells (for 2 or 6 h) at T = 0, T = 2, T = 4 and T = 6 days after induction of differentiation with ADM. After staining with Oil-red-O, lipids were quantified by spectrophotometric analysis at 540 mm following elution of cell-retained Oil-red-O with isopropanol. The amount of lipids in C2-RNase L cells at T = 0, before IPTG treatment, was set to 1. Error bars refer to the standard deviation obtained in three independent experimental points. (*) P<0.01 compared to untreated cells at the same time during adipocyte differentiation.

Article Snippet: After blocking with 10% (W/V) FCS in PBS, cells were incubated with a mouse monoclonal anti-Troponin T antibody (1/1000; Sigma) or a rabbit monoclonal anti-Fabp4/aP2 (1/100; Cell Signaling) at room temperature for 1 h. Cells were then washed and incubated with a donkey anti-mouse secondary antibody conjugated to TRITC or FITC (Santa-Cruz) or a donkey anti-rabbit secondary antibody conjugated to TRITC (Santa-Cruz) at room temperature for 1 h. For adipocyte differentiation, cells were cultured in adipocyte differentiating medium (ADM) [DMEM-10% (V/V) FCS, 5 µg/ml Bovine insulin (Sigma) and 1 µM dexamethazone (Sigma)] for 6 days.

Techniques: Binding Assay, Activity Assay, Autoradiography, Standard Deviation, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Quantitative RT-PCR, Staining, Incubation

Journal: PLoS ONE

Article Title: Endoribonuclease L (RNase L) Regulates the Myogenic and Adipogenic Potential of Myogenic Cells

doi: 10.1371/journal.pone.0007563

Figure Lengend Snippet: A: RNase L activity in C2-RNase L cells, C2-RNase L cells treated with IPTG and C2-RLI cells. C2-RLI cells (1, ), C2-RNase L cells (2, ) and C2-RNase L treated with 5 mM IPTG (3, ) for 6 h were analyzed for RNase L 2-5A binding activity with the 2-5A radiocovalent binding assay. Proteins were separated in 10% polyacrylamide gels. A representative autoradiography and a densitometry of the gel are shown. The amount of 2-5A binding activity in C2-RLI cells was set to 1. Error bars refer to the standard deviation obtained in three independent experiments. B: C2-RLI differentiation. Cells were fixed and incubated with Oil-red-O to reveal lipid accumulation (i = day 0, j = day 6 after induction of differentiation with ADM) and expression of Troponin T and Fabp4/aP2 was analyzed using monoclonal antibodies against Troponin T (Green, c,d) and Fabp4/aP2 (Red e,f) at day 0 (a,c,e,g,i) and day 6 after induction of differentiation with ADM (b,d,f,h,j). DNA was stained with Dapi (Blue a,b,g,h). A merge of Dapi, Troponin T and Fabp4/aP2 labeling is shown (g,h). Cells were observed at 20x, ( __ ): 20 µm. C: Quantification of lipids in C2-RLI cells at day 0 ( ) and day 6 after induction of differentiation with ADM ( ). A value of 1 corresponds to the amount of lipids in C2-RLI cells at T = 0, before differentiation. After staining with Oil-red-O, lipids were quantified by spectrophotometric analysis at 540 mm following elution of cell-retained Oil-red-O with isopropanol. Error bars refer to the standard deviation obtained in three independent experiments. (*) P<0.01 compared to untreated C2-RNase L cells and (**) P<0.01 compared to IPTG-treated C2-RNase L cells at the same time point during adipocyte differentiation. D: Analysis of mRNA expression by RT-PCR amplification at different time points during adipocyte differentiation. C2-RLI cells were switched to ADM to induce adipocyte differentiation at day 1. PCR products were analyzed by gel electrophoresis. Photographs of the gels are shown.

Article Snippet: After blocking with 10% (W/V) FCS in PBS, cells were incubated with a mouse monoclonal anti-Troponin T antibody (1/1000; Sigma) or a rabbit monoclonal anti-Fabp4/aP2 (1/100; Cell Signaling) at room temperature for 1 h. Cells were then washed and incubated with a donkey anti-mouse secondary antibody conjugated to TRITC or FITC (Santa-Cruz) or a donkey anti-rabbit secondary antibody conjugated to TRITC (Santa-Cruz) at room temperature for 1 h. For adipocyte differentiation, cells were cultured in adipocyte differentiating medium (ADM) [DMEM-10% (V/V) FCS, 5 µg/ml Bovine insulin (Sigma) and 1 µM dexamethazone (Sigma)] for 6 days.

Techniques: Activity Assay, Binding Assay, Autoradiography, Standard Deviation, Incubation, Expressing, Staining, Labeling, Reverse Transcription Polymerase Chain Reaction, Amplification, Nucleic Acid Electrophoresis