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

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