rabbit polyclonal antibody against the β-subunit of the insulin receptor sc-711  (Santa Cruz Biotechnology)

Journal: Journal of neurophysiology

Article Title: Brain Insulin Receptor Causes Activity-Dependent Current Suppression in the Olfactory Bulb Through Multiple Phosphorylation of Kv1.3

doi:

Figure Lengend Snippet: Activation of IR kinase induces suppression of Kv1.3. A: human embryonic kidney (HEK 293) cell cotransfected with Kv1.3 and insulin receptor kinase (IR) was voltage-clamped in the cell-attached configuration at –90 mV and stepped in 10-mV increments to 0 mV. Left, recorded using control bath solution. Middle, same cell after 20 min of bath-applied insulin (0.1 μg/ml). Right, cell-attached patch from a HEK 293 cell cotransfected with Kv1.3 and IR containing a truncated β subunit (IRtrunc; see methods) after an identical application of insulin under the same voltage protocol. For statistical comparisons, see Table 1. B: current–voltage plot of HEK 293 cells transfected as in A. Patches were voltage-clamped at –80 mV and stepped in 20-mV increments to +40 mV. ■ = Kv1.3 + IR control, ● = Kv1.3 + IR Insulin, ▲ = Kv1.3 + IR(trunc) Insulin. Inset: plot of the mean peak current magnitude of these patches as measured at the 140-mV depolarization step. Open bar, Kv1.3 + IR Control; solid bar, Kv1.3 + IR Insulin; crosshatched bar, Kv1.3 + IR(trunc) Insulin; *, significantly different paired t-test. C: HEK 293 cells cotransfected with Kv1.3 plus IR and sequentially double-labeled with Kv1.3 (1:200) and IRβ (1:100) as viewed at ×40 by confocal microscopy. No fluorescent signal is observed in the absence of primary antiserum. Arrows denote cells immunocytochemically labeled for both proteins, indicating the uptake of both cDNA constructs into single cells.

Article Snippet: Polyclonal antibody directed against the β subunit of the human insulin receptor was purchased from Upstate Biotechnology.

Techniques: Activation Assay, Control, Transfection, Labeling, Confocal Microscopy, Construct

Journal: PLoS ONE

Article Title: Insulin Activates Vagal Afferent Neurons Including those Innervating Pancreas via Insulin Cascade and Ca 2+ Influx: Its Dysfunction in IRS2-KO Mice with Hyperphagic Obesity

doi: 10.1371/journal.pone.0067198

Figure Lengend Snippet: A : Neurons immunoreactive to IR-β, revealed by Alexa 488 fluorescence, were observed in the section of mouse NG. The picture is representative of 13 sections from 4 mice. The right picture is the magnification of the area marked with dotted line in the left picture. B : mRNA expressions for IRs and IRS2 in NGs. Representative electrophoretic patterns of RT-PCR products of IR (left) and IRS2 (right) mRNAs in NGs. M = size maker, (−) = RT (−) as a negative control, (+) = RT (+). C∼E : Effects of insulin (10 −7 M), CCK (10 −8 M), and CAP (10 −7 M) on [Ca 2+ ] i in NG neurons derived from wild-type C57BL/6J ( C ) and IRS2 knockout mice ( D ). The [Ca 2+ ] i response to insulin, but not CCK-8 and CAP, were markedly attenuated in the NG neurons of IRS2 knockout mice ( D , n = 9) as compared with wild type ( C , n = 14). The amplitude of [Ca 2+ ] i response to each regent is normalized to that to KCl in each neuron and expressed as relative values (percentage) ( E ). The numbers on each bar indicate the numbers of neurons that responded to insulin, CCK-8 or CAP. The amplitude of [Ca 2+ ] i responses to insulin were suppressed by IRS2-deficiency. F∼H : Effects of a PI3K inhibitor LY294002 at 50 µM (LY, pretreatment for 1 hr, F ) and a MAPK inhibitor U0126 at 10 µM (U, pretreatment for 0.5 hr, G ) on insulin-induced [Ca 2+ ] i increases in NG neurons. The [Ca 2+ ] i traces of control experiments (C) are shown in Fig. 1E. In these three conditions, the amplitude of [Ca 2+ ] i response to each regent is normalized to that to KCl in each neuron and expressed as relative values (percentage) ( H ). The numbers on each bar indicate the numbers of neurons that responded insulin, CCK-8 or CAP. [Ca 2+ ] i responses to insulin were suppressed by LY but not U. * P <0.05, ** P <0.01 by paired t-test ( E ) and one-way ANOVA followed by Dunnett’s test vs. control ( H ).

Article Snippet: The sections were treated with blocking solution (2% normal goat serum and 2% BSA in PBS) for 30 min at RT, and then incubated with a rabbit polyclonal antibody against insulin receptor β-subunit (IR-β, sc-711, 1∶200, Santa cruz biotechnology, CA) or with a mouse monoclonal antibody against neurofilament (M0769, 1∶500, Dako, Denmark) for overnight at 4°C.

Techniques: Fluorescence, Reverse Transcription Polymerase Chain Reaction, Negative Control, Derivative Assay, Knock-Out, CCK-8 Assay, Control

Journal: PLoS ONE

Article Title: Insulin Activates Vagal Afferent Neurons Including those Innervating Pancreas via Insulin Cascade and Ca 2+ Influx: Its Dysfunction in IRS2-KO Mice with Hyperphagic Obesity

doi: 10.1371/journal.pone.0067198

Figure Lengend Snippet: A : Insulin concentrations in pancreatic vein (Panc. Vein) were higher than those in pancreatic artery (Panc. Artery) and portal vein (Portal) in fasted (left, n = 6) and ad lib fed rats (right, n = 10). Different letters above bars indicate significant difference, P <0.05 by one-way ANOVA followed by Tukey’s test. B : Immunofluorescence micrographs for IR-β (left; green), neurofilament (center; red), and both (right; merged). Immunoreactivity to IR-β was localized in the fibers/terminals of neurons that innervate pancreatic islets. Arrowheads show the neurons immunoreactive to both neurofilament and IR-β. C : Bright field (left) and DiI fluorescence (right) microphotographs indicate isolated NG neurons labeled (black arrow) and unlabeled (white arrow) with DiI injected into pancreas. D : A neuron pointed by black arrow in ( C ) responded to insulin at 10 −7 M with [Ca 2+ ] i increases. E : In total 625 neurons including labeled and unlabeled neurons, 46 neurons (7.4%) responded to insulin with [Ca 2+ ] i increases. DiI-labeled neurons responded to insulin with [Ca 2+ ] i increases with a much greater incidence (filled bar, 5 of 25 cells, 20.0%) than unlabeled neurons (dotted bar, 41 of 600 cells, 6.8%). * P <0.05 by chi-square test.

Article Snippet: The sections were treated with blocking solution (2% normal goat serum and 2% BSA in PBS) for 30 min at RT, and then incubated with a rabbit polyclonal antibody against insulin receptor β-subunit (IR-β, sc-711, 1∶200, Santa cruz biotechnology, CA) or with a mouse monoclonal antibody against neurofilament (M0769, 1∶500, Dako, Denmark) for overnight at 4°C.

Techniques: Immunofluorescence, Fluorescence, Isolation, Labeling, Injection