antibodies for total-irs2 mabs15  (Millipore)

Journal: Experimental Diabetes Research

Article Title: Insulin Receptor Substrate 2 Expression and Involvement in Neuronal Insulin Resistance in Diabetic Neuropathy

doi: 10.1155/2011/212571

Figure Lengend Snippet: IRS isoform expression in murine lumbar DRG. IRS isoforms were examined using RT-PCR ((a) and (b)) and Western blot ((c) and (d)). (a) RT-PCR was performed on adult C57Bl/6 mouse lumbar DRG ( n = 3 mice) and comparisons were made among IRS1, IRS2, IRS3, and IRS4. GAPDH was used as the housekeeping gene. ΔCt values for IRS2 were significantly lower than any other isoform. (b) Analysis of IRS mRNA levels that were normalized to IRS1 mRNA levels revealed that IRS2 mRNA expression was 27-fold higher than IRS1 in the DRG. *denotes P < .05 n = 3 mice. ((c) and (d)) Representative Western blots of IRS1 and IRS2 protein in mouse lumbar DRG. Equal amounts of protein (20 μ g) were loaded for each lane and samples were separated on 4–15% tris-glycine gel. Both IRS1 (c) and IRS2 (d) proteins were readily detectable. All mice were 8-week-old nondiabetic C57Bl/6 males.

Article Snippet: Primary antibodies were used at the following dilutions and incubations: total IRS2 (Millipore) 1 : 400 overnight at 4°C, mouse monoclonal Peripherin (Millipore) 1 : 2000 overnight at 4°C, and mouse monoclonal Neurofilament 200 (Sigma) 1 : 2000 overnight at 4°C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Experimental Diabetes Research

Article Title: Insulin Receptor Substrate 2 Expression and Involvement in Neuronal Insulin Resistance in Diabetic Neuropathy

doi: 10.1155/2011/212571

Figure Lengend Snippet: IRS2 protein expression pattern in murine lumbar DRG. Fluorescence immunohistochemistry was used to examine IRS2 expression in adult C57Bl/6 mouse lumbar DRG. ((a) and (d)) Photomicrographs of IRS2 immunoreactivity in DRG neurons. IRS2 was expressed in most neurons of the DRG in mice. ((b) and (e)) Photomicrographs of the same sections stained with antibodies to NF-200 (b), which labels large, myelinated neurons in the DRG or peripherin (e), which labels unmyelinated small DRG neurons. ((c) and (f)) Merged images of IRS2 and NF-200 (c) labeling illustrate that many IRS2-positive neurons also express NF-200, suggesting that many IRS2-positive neurons are large myelinated neurons. Similarly, merged images of IRS2 and peripherin (f) labeling illustrate that many IRS2-positive neurons coexpress peripherin, suggesting that many IRS2-positive neurons are small unmyelinated neurons. Scale bar = 100 μ m.

Article Snippet: Primary antibodies were used at the following dilutions and incubations: total IRS2 (Millipore) 1 : 400 overnight at 4°C, mouse monoclonal Peripherin (Millipore) 1 : 2000 overnight at 4°C, and mouse monoclonal Neurofilament 200 (Sigma) 1 : 2000 overnight at 4°C.

Techniques: Expressing, Fluorescence, Immunohistochemistry, Staining, Labeling

Journal: Experimental Diabetes Research

Article Title: Insulin Receptor Substrate 2 Expression and Involvement in Neuronal Insulin Resistance in Diabetic Neuropathy

doi: 10.1155/2011/212571

Figure Lengend Snippet: pSer(731)IRS2 is elevated in DRG neurons from type 1 and type 2 diabetic mice. Protein was harvested from adult mouse DRG culture from diabetic ob/ob and nondiabetic mice (a), freshly isolated DRG from STZ-injected diabetic and nondiabetic C57Bl/6 mice (b), and from DRG neurons grown in hyperglycemic and control conditions (c). Western blots were performed using antibodies that recognized phosphorylated ser731 resides on IRS2, and levels of Ser(731)IRS2 were normalized to total IRS2. (a) Comparisons of pSer(731)IRS2 levels in nondiabetic and diabetic ob/ob mice revealed a significant increase in pSer(731)IRS2 levels in diabetic mice. *denotes P < .05 versus nondiabetics. n = 6 for nondiabetic mice and n = 7 for diabetic mice. (b) Diabetes was induced in 8-week-old C57Bl/6 male mice with STZ, and diabetes was allowed to progress for 6 weeks. Similar to ob/ob diabetic mice, pSer(731)IRS2 levels were significantly elevated in STZ-injected diabetic mice. *denotes P < .05 versus nondiabetics. n = 5 for nondiabetic mice and n = 8 for diabetic mice. (c) DRG neurons from nondiabetic animals were grown in 10 mM (control) and 25 mM (hyperglycemic) glucose concentrations. There was no significant change in IRS2 serine phosphorylation levels between groups. n = 6 for 10 mM glucose and n = 7 for 25 mM glucose.

Article Snippet: Primary antibodies were used at the following dilutions and incubations: total IRS2 (Millipore) 1 : 400 overnight at 4°C, mouse monoclonal Peripherin (Millipore) 1 : 2000 overnight at 4°C, and mouse monoclonal Neurofilament 200 (Sigma) 1 : 2000 overnight at 4°C.

Techniques: Isolation, Injection, Western Blot

Journal: Experimental Diabetes Research

Article Title: Insulin Receptor Substrate 2 Expression and Involvement in Neuronal Insulin Resistance in Diabetic Neuropathy

doi: 10.1155/2011/212571

Figure Lengend Snippet: Total IRS2 and IR protein levels in mouse lumbar DRG. Protein was harvested from adult mouse DRG culture from diabetic ob/ob and nondiabetic mice. Western blots were performed using antibodies that recognized total IRS2 (a) or IR β subunit levels (b). In both cases protein levels were normalized to actin. (a) Total IRS2 levels were slightly decreased in diabetic ob/ob mice although this trend was not statistically significant ( P > .05). n = 7 for nondiabetic mice and n = 7 for diabetic mice. (b) IR β subunit protein levels were not statistically different between diabetic and nondiabetic mice ( P > .05). n = 5 for nondiabetic mice and n = 6 for diabetic mice.

Article Snippet: Primary antibodies were used at the following dilutions and incubations: total IRS2 (Millipore) 1 : 400 overnight at 4°C, mouse monoclonal Peripherin (Millipore) 1 : 2000 overnight at 4°C, and mouse monoclonal Neurofilament 200 (Sigma) 1 : 2000 overnight at 4°C.

Techniques: Western Blot

Journal: Experimental Diabetes Research

Article Title: Insulin Receptor Substrate 2 Expression and Involvement in Neuronal Insulin Resistance in Diabetic Neuropathy

doi: 10.1155/2011/212571

Figure Lengend Snippet: IRS isoform expression in murine lumbar DRG. IRS isoforms were examined using RT-PCR ((a) and (b)) and Western blot ((c) and (d)). (a) RT-PCR was performed on adult C57Bl/6 mouse lumbar DRG ( n = 3 mice) and comparisons were made among IRS1, IRS2, IRS3, and IRS4. GAPDH was used as the housekeeping gene. ΔCt values for IRS2 were significantly lower than any other isoform. (b) Analysis of IRS mRNA levels that were normalized to IRS1 mRNA levels revealed that IRS2 mRNA expression was 27-fold higher than IRS1 in the DRG. *denotes P < .05 n = 3 mice. ((c) and (d)) Representative Western blots of IRS1 and IRS2 protein in mouse lumbar DRG. Equal amounts of protein (20 μ g) were loaded for each lane and samples were separated on 4–15% tris-glycine gel. Both IRS1 (c) and IRS2 (d) proteins were readily detectable. All mice were 8-week-old nondiabetic C57Bl/6 males.

Article Snippet: Primary antibodies were used at the following dilutions and incubations: total IRS2 (Millipore) 1 : 400 overnight at 4°C, mouse monoclonal Peripherin (Millipore) 1 : 2000 overnight at 4°C, and mouse monoclonal Neurofilament 200 (Sigma) 1 : 2000 overnight at 4°C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Experimental Diabetes Research

Article Title: Insulin Receptor Substrate 2 Expression and Involvement in Neuronal Insulin Resistance in Diabetic Neuropathy

doi: 10.1155/2011/212571

Figure Lengend Snippet: IRS2 protein expression pattern in murine lumbar DRG. Fluorescence immunohistochemistry was used to examine IRS2 expression in adult C57Bl/6 mouse lumbar DRG. ((a) and (d)) Photomicrographs of IRS2 immunoreactivity in DRG neurons. IRS2 was expressed in most neurons of the DRG in mice. ((b) and (e)) Photomicrographs of the same sections stained with antibodies to NF-200 (b), which labels large, myelinated neurons in the DRG or peripherin (e), which labels unmyelinated small DRG neurons. ((c) and (f)) Merged images of IRS2 and NF-200 (c) labeling illustrate that many IRS2-positive neurons also express NF-200, suggesting that many IRS2-positive neurons are large myelinated neurons. Similarly, merged images of IRS2 and peripherin (f) labeling illustrate that many IRS2-positive neurons coexpress peripherin, suggesting that many IRS2-positive neurons are small unmyelinated neurons. Scale bar = 100 μ m.

Article Snippet: Primary antibodies were used at the following dilutions and incubations: total IRS2 (Millipore) 1 : 400 overnight at 4°C, mouse monoclonal Peripherin (Millipore) 1 : 2000 overnight at 4°C, and mouse monoclonal Neurofilament 200 (Sigma) 1 : 2000 overnight at 4°C.

Techniques: Expressing, Fluorescence, Immunohistochemistry, Staining, Labeling

Journal: Experimental Diabetes Research

Article Title: Insulin Receptor Substrate 2 Expression and Involvement in Neuronal Insulin Resistance in Diabetic Neuropathy

doi: 10.1155/2011/212571

Figure Lengend Snippet: pSer(731)IRS2 is elevated in DRG neurons from type 1 and type 2 diabetic mice. Protein was harvested from adult mouse DRG culture from diabetic ob/ob and nondiabetic mice (a), freshly isolated DRG from STZ-injected diabetic and nondiabetic C57Bl/6 mice (b), and from DRG neurons grown in hyperglycemic and control conditions (c). Western blots were performed using antibodies that recognized phosphorylated ser731 resides on IRS2, and levels of Ser(731)IRS2 were normalized to total IRS2. (a) Comparisons of pSer(731)IRS2 levels in nondiabetic and diabetic ob/ob mice revealed a significant increase in pSer(731)IRS2 levels in diabetic mice. *denotes P < .05 versus nondiabetics. n = 6 for nondiabetic mice and n = 7 for diabetic mice. (b) Diabetes was induced in 8-week-old C57Bl/6 male mice with STZ, and diabetes was allowed to progress for 6 weeks. Similar to ob/ob diabetic mice, pSer(731)IRS2 levels were significantly elevated in STZ-injected diabetic mice. *denotes P < .05 versus nondiabetics. n = 5 for nondiabetic mice and n = 8 for diabetic mice. (c) DRG neurons from nondiabetic animals were grown in 10 mM (control) and 25 mM (hyperglycemic) glucose concentrations. There was no significant change in IRS2 serine phosphorylation levels between groups. n = 6 for 10 mM glucose and n = 7 for 25 mM glucose.

Article Snippet: Primary antibodies were used at the following dilutions and incubations: total IRS2 (Millipore) 1 : 400 overnight at 4°C, mouse monoclonal Peripherin (Millipore) 1 : 2000 overnight at 4°C, and mouse monoclonal Neurofilament 200 (Sigma) 1 : 2000 overnight at 4°C.

Techniques: Isolation, Injection, Control, Western Blot, Phospho-proteomics

Journal: Experimental Diabetes Research

Article Title: Insulin Receptor Substrate 2 Expression and Involvement in Neuronal Insulin Resistance in Diabetic Neuropathy

doi: 10.1155/2011/212571

Figure Lengend Snippet: Total IRS2 and IR protein levels in mouse lumbar DRG. Protein was harvested from adult mouse DRG culture from diabetic ob/ob and nondiabetic mice. Western blots were performed using antibodies that recognized total IRS2 (a) or IR β subunit levels (b). In both cases protein levels were normalized to actin. (a) Total IRS2 levels were slightly decreased in diabetic ob/ob mice although this trend was not statistically significant ( P > .05). n = 7 for nondiabetic mice and n = 7 for diabetic mice. (b) IR β subunit protein levels were not statistically different between diabetic and nondiabetic mice ( P > .05). n = 5 for nondiabetic mice and n = 6 for diabetic mice.

Article Snippet: Primary antibodies were used at the following dilutions and incubations: total IRS2 (Millipore) 1 : 400 overnight at 4°C, mouse monoclonal Peripherin (Millipore) 1 : 2000 overnight at 4°C, and mouse monoclonal Neurofilament 200 (Sigma) 1 : 2000 overnight at 4°C.

Techniques: Western Blot