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Fluorescein (FITC)-AffiniPure Donkey Anti-Rabbit IgG (H+L) (min X Bov,Ck,Gt,GP,Sy Hms,Hrs,Hu,Ms,Rat,Shp Sr Prot) antibody


Antibody ID


Target Antigen

Rabbit IgG (H+L)

Proper Citation

(Jackson ImmunoResearch Labs Cat# 711-095-152, RRID:AB_2315776)


polyclonal antibody


Originating manufacturer of this product


Jackson ImmunoResearch Labs Go To Vendor

Cat Num


Synaptotagmin 4 Regulates Pancreatic β Cell Maturation by Modulating the Ca2+ Sensitivity of Insulin Secretion Vesicles.

  • Huang C
  • Dev. Cell
  • 2018 May 7

Literature context:


Islet β cells from newborn mammals exhibit high basal insulin secretion and poor glucose-stimulated insulin secretion (GSIS). Here we show that β cells of newborns secrete more insulin than adults in response to similar intracellular Ca2+ concentrations, suggesting differences in the Ca2+ sensitivity of insulin secretion. Synaptotagmin 4 (Syt4), a non-Ca2+ binding paralog of the β cell Ca2+ sensor Syt7, increased by ∼8-fold during β cell maturation. Syt4 ablation increased basal insulin secretion and compromised GSIS. Precocious Syt4 expression repressed basal insulin secretion but also impaired islet morphogenesis and GSIS. Syt4 was localized on insulin granules and Syt4 levels inversely related to the number of readily releasable vesicles. Thus, transcriptional regulation of Syt4 affects insulin secretion; Syt4 expression is regulated in part by Myt transcription factors, which repress Syt4 transcription. Finally, human SYT4 regulated GSIS in EndoC-βH1 cells, a human β cell line. These findings reveal the role that altered Ca2+ sensing plays in regulating β cell maturation.

Funding information:
  • Cancer Research UK - 12183(United Kingdom)
  • NIDDK NIH HHS - R01 DK050203()
  • NIDDK NIH HHS - R01 DK090570()

Activation of the Notch Signaling Pathway In Vivo Elicits Changes in CSL Nuclear Dynamics.

  • Gomez-Lamarca MJ
  • Dev. Cell
  • 2018 Mar 12

Literature context:


A key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response-in the absence of any amplification step-remains to be elucidated. To address this question, we developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or "assisted" loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which we propose confer sensitivity to small input changes and facilitate timely shut-down.

Funding information:
  • Wellcome Trust - CA084179(United Kingdom)
  • Wellcome Trust - R01 CA178974()

Nicotine increases colon cancer cell migration and invasion through epithelial to mesenchymal transition (EMT): COX-2 involvement.

  • Dinicola S
  • J. Cell. Physiol.
  • 2017 Dec 8

Literature context:


Cigarette smoking is a recognized risk factor for colon cancer and nicotine, the principal active component of tobacco, plays a pivotal role in increasing colon cancer cell growth and survival. The aim of this study was to determine the effect of nicotine on cellular Caco-2 and HCT-8 migration and invasion, focusing on epithelial to mesenchymal transition (EMT) induction, and COX-2 pathway involvement. In both these cell lines, treatment with nicotine increased COX-2 expression and the release of its enzymatic product PGE2 . Moreover, nicotine-stimulated cells showed increased migratory and invasive behavior, mesenchymal markers up-regulation and epithelial markers down-regulation, nuclear translocation of the β-catenin, increase of MMP-2 and MMP-9 activity, and enhanced NF-κB expression. Noticeably, all these effects are largely mediated by COX-2 activity, as simultaneous treatment of both cell lines with nicotine and NS-398, a selective COX-2 inhibitor, greatly reduced the number of migrating and invading cells and reverted nicotine-induced EMT. These findings emphasize that nicotine triggers EMT, leading hence to increased migration and invasiveness of colon cancer cells. Thereby, the use of COX-2 inhibitor drugs might likely counteract nicotine-mediated EMT effects on colon cancer development and progression.

Funding information:
  • NCI NIH HHS - R01 CA119018-04(United States)

VGLUT1 synapses and P-boutons on regenerating motoneurons after nerve crush.

  • Schultz AJ
  • J. Comp. Neurol.
  • 2017 Sep 1

Literature context:


Stretch-sensitive Ia afferent monosynaptic connections with motoneurons form the stretch reflex circuit. After nerve transection, Ia afferent synapses and stretch reflexes are permanently lost, even after regeneration and reinnervation of muscle by motor and sensory afferents is completed in the periphery. This loss greatly affects full recovery of motor function. However, after nerve crush, reflex muscle forces during stretch do recover after muscle reinnervation and reportedly exceed 140% baseline values. This difference might be explained by structural preservation after crush of Ia afferent synapses on regenerating motoneurons and decreased presynaptic inhibitory control. We tested these possibilities in rats after crushing the tibial nerve (TN), and using Vesicular GLUtamate Transporter 1 (VGLUT1) and the 65 kDa isoform of glutamic acid-decarboxylase (GAD65) as markers of, respectively, Ia afferent synapses and presynaptic inhibition (P-boutons) on retrogradely labeled motoneurons. We analyzed motoneurons during regeneration (21 days post crush) and after they reinnervate muscle (3 months). The results demonstrate a significant loss of VGLUT1 terminals on dendrites and cell bodies at both 21 days and 3 months post-crush. However, in both cellular compartments, the reductions were small compared to those observed after TN full transection. In addition, we found a significant decrease in the number of GAD65 P-boutons per VGLUT1 terminal and their coverage of VGLUT1 boutons. The results support the hypothesis that better preservation of Ia afferent synapses and a change in presynaptic inhibition could contribute to maintain or even increase the stretch reflex after nerve crush and by difference to nerve transection.

Funding information:
  • NINDS NIH HHS - F31 NS095528()
  • NINDS NIH HHS - P01 NS057228()

Glycinergic Input to the Mouse Basal Forebrain Cholinergic Neurons.

  • Bardóczi Z
  • J. Neurosci.
  • 2017 Sep 27

Literature context:


The basal forebrain (BF) receives afferents from brainstem ascending pathways, which has been implicated first by Moruzzi and Magoun (1949) to induce forebrain activation and cortical arousal/waking behavior; however, it is very little known about how brainstem inhibitory inputs affect cholinergic functions. In the current study, glycine, a major inhibitory neurotransmitter of brainstem neurons, and gliotransmitter of local glial cells, was tested for potential interaction with BF cholinergic (BFC) neurons in male mice. In the BF, glycine receptor α subunit-immunoreactive (IR) sites were localized in choline acetyltransferase (ChAT)-IR neurons. The effect of glycine on BFC neurons was demonstrated by bicuculline-resistant, strychnine-sensitive spontaneous IPSCs (sIPSCs; 0.81 ± 0.25 × 10-1 Hz) recorded in whole-cell conditions. Potential neuronal as well as glial sources of glycine were indicated in the extracellular space of cholinergic neurons by glycine transporter type 1 (GLYT1)- and GLYT2-IR processes found in apposition to ChAT-IR cells. Ultrastructural analyses identified synapses of GLYT2-positive axon terminals on ChAT-IR neurons, as well as GLYT1-positive astroglial processes, which were localized in the vicinity of synapses of ChAT-IR neurons. The brainstem raphe magnus was determined to be a major source of glycinergic axons traced retrogradely from the BF. Our results indicate a direct effect of glycine on BFC neurons. Furthermore, the presence of high levels of plasma membrane glycine transporters in the vicinity of cholinergic neurons suggests a tight control of extracellular glycine in the BF.SIGNIFICANCE STATEMENT Basal forebrain cholinergic (BFC) neurons receive various activating inputs from specific brainstem areas and channel this information to the cortex via multiple projections. So far, very little is known about inhibitory brainstem afferents to the BF. The current study established glycine as a major regulator of BFC neurons by (1) identifying glycinergic neurons in the brainstem projecting to the BF, (2) showing glycine receptor α subunit-immunoreactive (IR) sites in choline acetyltransferase (ChAT)-IR neurons, (3) demonstrating glycine transporter type 2 (GLYT2)-positive axon terminals synapsing on ChAT-IR neurons, and (4) localizing GLYT1-positive astroglial processes in the vicinity of synapses of ChAT-IR neurons. The effect of glycine on BFC neurons was demonstrated by bicuculline-resistant, strychnine-sensitive spontaneous IPSCs recorded in whole-cell conditions.

Wfs1- deficient rats develop primary symptoms of Wolfram syndrome: insulin-dependent diabetes, optic nerve atrophy and medullary degeneration.

  • Plaas M
  • Sci Rep
  • 2017 Aug 31

Literature context:


Wolfram syndrome (WS) is a rare autosomal-recessive disorder that is caused by mutations in the WFS1 gene and is characterized by juvenile-onset diabetes, optic atrophy, hearing loss and a number of other complications. Here, we describe the creation and phenotype of Wfs1 mutant rats, in which exon 5 of the Wfs1 gene is deleted, resulting in a loss of 27 amino acids from the WFS1 protein sequence. These Wfs1-ex5-KO232 rats show progressive glucose intolerance, which culminates in the development of diabetes mellitus, glycosuria, hyperglycaemia and severe body weight loss by 12 months of age. Beta cell mass is reduced in older mutant rats, which is accompanied by decreased glucose-stimulated insulin secretion from 3 months of age. Medullary volume is decreased in older Wfs1-ex5-KO232 rats, with the largest decreases at the level of the inferior olive. Finally, older Wfs1-ex5-KO232 rats show retinal gliosis and optic nerve atrophy at 15 months of age. Electron microscopy revealed axonal degeneration and disorganization of the myelin in the optic nerves of older Wfs1-ex5-KO232 rats. The phenotype of Wfs1-ex5-KO232 rats indicates that they have the core symptoms of WS. Therefore, we present a novel rat model of WS.

Selective synaptic connections in the retinal pathway for night vision.

  • Beaudoin DL
  • J. Comp. Neurol.
  • 2017 Aug 30

Literature context:


The mammalian retina encodes visual information in dim light using rod photoreceptors and a specialized circuit: rods→rod bipolar cells→AII amacrine cell. The AII amacrine cell uses sign-conserving electrical synapses to modulate ON cone bipolar cell terminals and sign-inverting chemical (glycinergic) synapses to modulate OFF cone cell bipolar terminals; these ON and OFF cone bipolar terminals then drive the output neurons, retinal ganglion cells (RGCs), following light increments and decrements, respectively. The AII amacrine cell also makes direct glycinergic synapses with certain RGCs, but it is not well established how many types receive this direct AII input. Here, we investigated functional AII amacrine→RGC synaptic connections in the retina of the guinea pig (Cavia porcellus) by recording inhibitory currents from RGCs in the presence of ionotropic glutamate receptor (iGluR) antagonists. This condition isolates a specific pathway through the AII amacrine cell that does not require iGluRs: cone→ON cone bipolar cell→AII amacrine cell→RGC. These recordings show that AII amacrine cells make direct synapses with OFF Alpha, OFF Delta and a smaller OFF transient RGC type that co-stratifies with OFF Alpha cells. However, AII amacrine cells avoid making synapses with numerous RGC types that co-stratify with the connected RGCs. Selective AII connections ensure that a privileged minority of RGC types receives direct input from the night-vision pathway, independent from OFF bipolar cell activity. Furthermore, these results illustrate the specificity of retinal connections, which cannot be predicted solely by co-stratification of dendrites and axons within the inner plexiform layer.

A Peptidergic Circuit Links the Circadian Clock to Locomotor Activity.

  • King AN
  • Curr. Biol.
  • 2017 Jul 10

Literature context:


The mechanisms by which clock neurons in the Drosophila brain confer an ∼24-hr rhythm onto locomotor activity are unclear, but involve the neuropeptide diuretic hormone 44 (DH44), an ortholog of corticotropin-releasing factor. Here we identified DH44 receptor 1 as the relevant receptor for rest:activity rhythms and mapped its site of action to hugin-expressing neurons in the subesophageal zone (SEZ). We traced a circuit that extends from Dh44-expressing neurons in the pars intercerebralis (PI) through hugin+ SEZ neurons to the ventral nerve cord. Hugin neuropeptide, a neuromedin U ortholog, also regulates behavioral rhythms. The DH44 PI-Hugin SEZ circuit controls circadian locomotor activity in a daily cycle but has minimal effect on feeding rhythms, suggesting that the circadian drive to feed can be separated from circadian locomotion. These findings define a linear peptidergic circuit that links the clock to motor outputs to modulate circadian control of locomotor activity.

Llgl1 Connects Cell Polarity with Cell-Cell Adhesion in Embryonic Neural Stem Cells.

  • Jossin Y
  • Dev. Cell
  • 2017 Jun 5

Literature context:


Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. We report here that mice with embryonic neural stem-cell-specific deletion of Llgl1 (Nestin-Cre/Llgl1fl/fl), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1fl/fl brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. We show that LLGL1 directly binds to and promotes internalization of N-cadherin, and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1, restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells.

Funding information:
  • NCI NIH HHS - R01 CA131047()
  • NCI NIH HHS - R01 CA179914()
  • NICHD NIH HHS - T32 HD007183()
  • NINDS NIH HHS - R01 NS080194()

TCF7L1 promotes skin tumorigenesis independently of β-catenin through induction of LCN2.

  • Ku AT
  • Elife
  • 2017 May 3

Literature context:


The transcription factor TCF7L1 is an embryonic stem cell signature gene that is upregulated in multiple aggressive cancer types, but its role in skin tumorigenesis has not yet been defined. Here we document TCF7L1 upregulation in skin squamous cell carcinoma (SCC) and demonstrate that TCF7L1 overexpression increases tumor incidence, tumor multiplicity, and malignant progression in the chemically induced mouse model of skin SCC. Additionally, we show that downregulation of TCF7L1 and its paralogue TCF7L2 reduces tumor growth in a xenograft model of human skin SCC. Using separation-of-function mutants, we show that TCF7L1 promotes tumor growth, enhances cell migration, and overrides oncogenic RAS-induced senescence independently of its interaction with β-catenin. Through transcriptome profiling and combined gain- and loss-of-function studies, we identified LCN2 as a major downstream effector of TCF7L1 that drives tumor growth. Our findings establish a tumor-promoting role for TCF7L1 in skin and elucidate the mechanisms underlying its tumorigenic capacity.

Funding information:
  • NCI NIH HHS - P30 CA016672()
  • NCI NIH HHS - P30 CA125123()
  • NCI NIH HHS - R01 CA194062()
  • NCI NIH HHS - R01 CA194617()
  • NCRR NIH HHS - S10 RR024574()
  • NHLBI NIH HHS - T32 HL092332()
  • NIAID NIH HHS - P30 AI036211()
  • NIDCD NIH HHS - R01 DC00189.(United States)
  • NIGMS NIH HHS - T32 GM088129()

RhoD Inhibits RhoC-ROCK-Dependent Cell Contraction via PAK6.

  • Durkin CH
  • Dev. Cell
  • 2017 May 8

Literature context:


RhoA-mediated regulation of myosin-II activity in the actin cortex controls the ability of cells to contract and bleb during a variety of cellular processes, including cell migration and division. Cell contraction and blebbing also frequently occur as part of the cytopathic effect seen during many different viral infections. We now demonstrate that the vaccinia virus protein F11, which localizes to the plasma membrane, is required for ROCK-mediated cell contraction from 2 hr post infection. Curiously, F11-induced cell contraction is dependent on RhoC and not RhoA signaling to ROCK. Moreover, RhoC-driven cell contraction depends on the upstream inhibition of RhoD signaling by F11. This inhibition prevents RhoD from regulating its downstream effector Pak6, alleviating the suppression of RhoC by the kinase. Our observations with vaccinia have now demonstrated that RhoD recruits Pak6 to the plasma membrane to antagonize RhoC signaling during cell contraction and blebbing.

Delayed Accumulation of H3K27me3 on Nascent DNA Is Essential for Recruitment of Transcription Factors at Early Stages of Stem Cell Differentiation.

  • Petruk S
  • Mol. Cell
  • 2017 Apr 20

Literature context:


Recruitment of transcription factors (TFs) to repressed genes in euchromatin is essential to activate new transcriptional programs during cell differentiation. However, recruitment of all TFs, including pioneer factors, is impeded by condensed H3K27me3-containing chromatin. Single-cell and gene-specific analyses revealed that, during the first hours of induction of differentiation of mammalian embryonic stem cells (ESCs), accumulation of the repressive histone mark H3K27me3 is delayed after DNA replication, indicative of a decondensed chromatin structure in all regions of the replicating genome. This delay provides a critical "window of opportunity" for recruitment of lineage-specific TFs to DNA. Increasing the levels of post-replicative H3K27me3 or preventing S phase entry inhibited recruitment of new TFs to DNA and significantly blocked cell differentiation. These findings suggest that recruitment of lineage-specifying TFs occurs soon after replication and is facilitated by a decondensed chromatin structure. This insight may explain the developmental plasticity of stem cells and facilitate their exploitation for therapeutic purposes.

Funding information:
  • NCI NIH HHS - R01 CA164834()
  • NHLBI NIH HHS - R01 HL127895()
  • NIAID NIH HHS - R01 AI125650()
  • NIGMS NIH HHS - R01 GM075141()
  • NINDS NIH HHS - R01 NS075839()

α3 Chains of type V collagen regulate breast tumour growth via glypican-1.

  • Huang G
  • Nat Commun
  • 2017 Jan 19

Literature context:


Pericellular α3(V) collagen can affect the functioning of cells, such as adipocytes and pancreatic β cells. Here we show that α3(V) chains are an abundant product of normal mammary gland basal cells, and that α3(V) ablation in a mouse mammary tumour model inhibits mammary tumour progression by reducing the proliferative potential of tumour cells. These effects are shown to be primarily cell autonomous, from loss of α3(V) chains normally produced by tumour cells, in which they affect growth by enhancing the ability of cell surface proteoglycan glypican-1 to act as a co-receptor for FGF2. Thus, a mechanism is presented for microenvironmental influence on tumour growth. α3(V) chains are produced in both basal-like and luminal human breast tumours, and its expression levels are tightly coupled with those of glypican-1 across breast cancer types. Evidence indicates α3(V) chains as potential targets for inhibiting tumour growth and as markers of oncogenic transformation.

Structural basis for serotonergic regulation of neural circuits in the mouse olfactory bulb.

  • Suzuki Y
  • J. Comp. Neurol.
  • 2015 Feb 1

Literature context:


Olfactory processing is well known to be regulated by centrifugal afferents from other brain regions, such as noradrenergic, acetylcholinergic, and serotonergic neurons. Serotonergic neurons widely innervate and regulate the functions of various brain regions. In the present study, we focused on serotonergic regulation of the olfactory bulb (OB), one of the most structurally and functionally well-defined brain regions. Visualization of a single neuron among abundant and dense fibers is essential to characterize and understand neuronal circuits. We accomplished this visualization by successfully labeling and reconstructing serotonin (5-hydroxytryptamine: 5-HT) neurons by infection with sindbis and adeno-associated virus into dorsal raphe nuclei (DRN) of mice. 5-HT synapses were analyzed by correlative confocal laser microscopy and serial-electron microscopy (EM) study. To further characterize 5-HT neuronal and network function, we analyzed whether glutamate was released from 5-HT synaptic terminals using immuno-EM. Our results are the first visualizations of complete 5-HT neurons and fibers projecting from DRN to the OB with bifurcations. We found that a single 5-HT axon can form synaptic contacts to both type 1 and 2 periglomerular cells within a single glomerulus. Through immunolabeling, we also identified vesicular glutamate transporter 3 in 5-HT neurons terminals, indicating possible glutamatergic transmission. Our present study strongly implicates the involvement of brain regions such as the DRN in regulation of the elaborate mechanisms of olfactory processing. We further provide a structure basis of the network for coordinating or linking olfactory encoding with other neural systems, with special attention to serotonergic regulation.

Funding information:
  • NEI NIH HHS - EY020826(United States)