Journal: Journal of neuroendocrinology

Article Title: Annexin A1 Complex Mediates Oxytocin Vesicle Transport

doi: 10.1111/jne.12112

Figure Lengend Snippet: ANXA1, AKAP150, and microtubule motors form a complex in mouse hypothalamus and N11 cells. The cytosols (5 mg/mL) of mouse hypothalamus tissues (A) or N11 cells (B–D) that were treated with DMSO (B), 10 μM forskolin (C, Forsk), or 10 nM PMA (D) were mixed with the antibody 4660 along with protein A beads (60 μL) at 4°C for 24 h. Proteins pulled down by anti-ANXA1 antibody (αAN) were probed by immunoblotting. (E) The cytosols (5 mg/mL, post-nuclear supernatant [SN]) of N11 cells were mixed with anti-p150 (dynactin) antibody for co-immunoprecipitation. Proteins bound to dynactin were detected by immunoblotting.

Article Snippet: Antibodies to ANXA1 (sc-12740), AKAP150 (sc-10765), dynein intermediate chain (DIC, sc-13524), kinesin-1 (kinesin heavy chain, sc-28538), kinesin-2 (KIF3A, sc-135960), CPE (sc-136252), dynactin (p150, sc-11363) and syntaxin-1 (sc-12736) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Techniques: Western Blot, Immunoprecipitation

Journal: Journal of neuroendocrinology

Article Title: Annexin A1 Complex Mediates Oxytocin Vesicle Transport

doi: 10.1111/jne.12112

Figure Lengend Snippet: Schematic illustration of how PKA and PKC affect the interaction of ANXA1 with AKAP150 and kinesin-2, thus changing the direction of movement of oxytocin-containing vesicles. (A) Upon activation of PKA that consists of two catalytic [C] and two regulatory [R] subunits, the regulated subunits withdraw their inhibitory action from the catalytic subunits. Then, the catalytic subunits increase the binding of kinesin-2 and AKAP150 to ANXA1 on light-density oxytocin (OXT) vesicle without affecting the interaction of cytoplasmic dynein-dynactin with ANXA1, resulting in enhancement of anterograde transport of the vesicles to the axon terminal. (B) Activated PKC inhibits the interaction of kinesin-2 with AKAP150-ANXA1 complex without affecting the interaction of cytoplasmic dynein-dynactin with ANXA1 complex, resulting in inhibition of anterograde transport of oxytocin vesicles to the axon terminal. (C) Activation of PKA enhances the anterograde transport of light-density oxytocin vesicles to the axon terminals while activation of PKC inhibits the anterograde transport. Activation of PKC increases somatic exocytosis of oxytocin vesicles. Some heavy density oxytocin vesicles are transported to the axon and dendrites.

Article Snippet: Antibodies to ANXA1 (sc-12740), AKAP150 (sc-10765), dynein intermediate chain (DIC, sc-13524), kinesin-1 (kinesin heavy chain, sc-28538), kinesin-2 (KIF3A, sc-135960), CPE (sc-136252), dynactin (p150, sc-11363) and syntaxin-1 (sc-12736) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Techniques: Activation Assay, Binding Assay, Inhibition

Journal: Journal of Neuroscience

Article Title: Dynamic Changes in Presynaptic and Axonal Transport Proteins Combined with Striatal Neuroinflammation Precede Dopaminergic Neuronal Loss in a Rat Model of AAV -Synucleinopathy

doi: 10.1523/jneurosci.5427-08.2009

Figure Lengend Snippet: Figure3. Striatalchangesinthelevelsofproteinsinvolvedinsynaptictransmissionandaxonaltransportat8weekspostAAV -synuclein injection. Western blot was performed using membrane fraction (P2) of striatal lysate to measure levels of proteins involvedinsynaptictransmission.Amongtheseproteins,rabphilin3Aandsyntaxinlevelswerereduced(A,B).Westernblotwas performed using total lysate (T) of the striatum to measure levels of protein involved in axonal transport. Levels of anterograde transport motor proteins including KIF1A, KIF1B, KIF2A, KIF3A and myosin Va were markedly decreased, whereas levels of retrograde transport motor proteins including dynein, dynamitin and dynactin1 were dramatically increased (C, D). Levels of cytoskeletalproteinssuchasactin,neurofilaments,-tubulinand-tubulin,werealtered(C,D).Opticaldensitiesoftheindivid- ualbandswerequantifiedusingNIHImageJ.Opticaldensitiesof-synucleinoverexpressingconditionswerenormalizedbythe averagedvalueofGFPexpressingcondition.DataareshownasmeanSEM(AAVGFP,n46;AAVA53T-synuclein,n 46; *p 0.05 two tail t test).

Article Snippet: After at least 1 h of blocking in 5% nonfat dry milk, the membranes were incubated overnight at 4°C in various primary antibodies [ -synuclein (clone 42, BD Transduction Laboratories; 1:2000), GFP (Invitrogen; 1:5000) TH (Pel Freeze; 1:3000), dopamine transporter (DAT, Millipore Bioscience Research Reagents; 1:2000), Vesicular Monoamine Transporter 2 (VMAT2, Pel Freeze; 1:1000), SNAP-25 (Millipore Bioscience Research Reagents; 1:4000), Rabphilin3A (BD Transduction Laboratories; 1:2000), RAB3A (Affinity Bioreagent; 1:3000), syntaxin (Millipore Bioscience Research Reagents; 1:2000), synaptophysin (Santa Cruz; 1:500), synaptotagmin (BD Transduction Laboratories; 1:5000), synapsin (Millipore Bioscience Research Reagents; 1:5000), Munc-18 (Affinity Bioreagents; 1:3000), KIF1A (clone 16, BD Transduction Laboratories; 1:2000), KIF1B (Bethyl Laboratory; 1:2000), KIF2A (Abcam; 1:10,000), KIF3A (Abcam; 1:2000), KIF5 (Abcam; 1:1000), KIF17 (Abcam; 1:500), myosin Va (Sigma; 1:500), dynein (clone 74.1, Millipore Bioscience Research Reagents; 1:2000), dynamitin (Millipore Bioscience Research Reagents; 1:2000), dynactin1 (Novus,1:1000), -tubulin (clone DM1A, Upstate; 1:5000), -tubulin (Upstate; 1:5000), -tubulin (clone GTU-88, Sigma; 1:5000), Actin (clone C-2, Santa Cruz; 1:500), neurofilament 160 (clone NN18, Sigma; 1:1000), neurofilament 200 (clone N52, Sigma; 1:1000), and GAPDH (Millipore Bioscience Research Reagents; 1:5000)].

Techniques: Injection, Western Blot, Membrane

Journal: Kidney360

Article Title: Dynein-Mediated Trafficking: A New Mechanism of Diabetic Podocytopathy

doi: 10.34067/KID.0006852022

Figure Lengend Snippet: Antibodies and fluorescent probes (immunofluorescent, Western blotting, immunoprecipitation]

Article Snippet: Table 2 Antibody Dilution Company Cat# Mouse antinephrin (G-8) IF: 1:100 Santa Cruz sc-376522 Mouse antinephrin conjugated to agarose IP: 2 μg/400 μl lysate Santa Cruz sc-376522 AC Normal mouse IgG conjugated to agarose IP: 2 μg/400 μl lysate Santa Cruz sc-2343 Rabbit antinephrin WB: 1:1000 Invitrogen PA5-91907 Sheep anti-DCTN1 WB: 1:1000 IF: 1:100 R&D Systems AF6657 Mouse anti-Dynll1 IF: 1:100 Santa Cruz sc-136287 Rabbit anti-Dynll1 WB: 1:1000 IF: 1:100 Thermo Fisher PA5-97920 Rabbit anti-INF2 IF: 1:100 Bethyl Lab A303-427A Rabbit anti-HDAC6 WB: 1:1000 Novus NBP1-78981 Donkey anti-mouse (Alexa Fluor 488) IF: 1:200 Thermo Fisher A32766 Donkey anti-rabbit (Alexa Fluor 488) IF: 1:200 Thermo Fisher A32790 Donkey anti-mouse (Alexa Fluor 594) IF: 1:200 Thermo Fisher A32744 Donkey anti-sheep (Alexa Fluor 488) IF: 1:200 Thermo Fisher A-11015 Mouse anti-rabbit IgG-HRP WB: 1:5000 Santa Cruz sc-2357 Rabbit anti-sheep IgG-HRP WB: 1:5000 Thermo Fisher 31480 Biotin mouse anti-ubiquitin WB: 1:1000 Invitrogen 13-6078-82 Mouse anti β-actin-HRP WB: 1:5000 Santa Cruz sc-47778 Mouse anti-WT1 IF: 1:100 Novus NBP24460700 Mouse anti-acetylated α-tubulin IF: 1:200 Santa Cruz sc-23950 Acti-stain 488 phalloidin Cytoskeleton PHDG1-A ViaFluor microtubule stains (488) Biotium 70062 Open in a separate window IF, immunofluorescent; IP, immunoprecipitation; WB, Western blotting; INF2, inverted formin 2; HDAC6, histone deacetylase 6; HRP, horseradish peroxidase.

Techniques: Western Blot, Immunoprecipitation

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: TaqMan probes used in the studies

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques:

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: Expression of dynactin DCTN1 and DCTN3 mRNA in the CNS of SALS and control cases. The expression was studied by real-time qPCR, as described in the Material and method section. The results were quantified as the ratio of studied dynactin (DCTN1 or DCTN3) expression to the expression of housekeeping genes (B2M and GusB; ΔCt method). No. 1–5, cases with SALS; a – e , control cases. Black bars motor cortex; striped bars sensory cortex

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques: Expressing, Control

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: Mean dynactin subunits expression in various parts of human CNS

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques: Expressing, Control

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: Expression of dynactin DCTN1 and DCTN3 protein in the CNS of SALS and control cases. The expression was studied by Western blotting, as indicated in the Material and method section. Comparable amounts of protein (20 μg for DCTN1 and 40 μg for DCTN3) from representative control (no D) and SALS (no 4) brains were run in each lane. A; DCTN1: line 1 control motor cortex (optical density: OD 651), line 2 control sensory cortex (OD 847), line 3 SALS motor cortex (OD 439), line 4 SALS sensory cortex (OD 519), line 5 control spinal cord (OD 940), line 6 SALS spinal cord (OD 627). b DCTN3: line 1 control motor cortex (OD 211), line 2 control sensory cortex (OD 238), line 3 SALS motor cortex (OD 210), line 4 SALS sensory cortex (OD 290), line 5 control spinal cord (OD 180), line 6 SALS spinal cord (OD 92)

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques: Expressing, Control, Western Blot

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: Representative immunohistochemistry for dynactin DCTN1 on sections from SALS and control human cases. a Immunopossitive neurons and their processes in SALS motor cortex and visible moderate loss of neuronal cells; b positive immunoreactivity in SALS neurons and axons in the sensory cortex; c moderate immunoexpression in pericaria and axons of the preserved SALS anterior horn motoneurons with features of degeneration; d pronounced immunoreactivity of control neuronal processes and pericaria in the motor cortex; e positive immune reaction of different intensity in control axons and neuronal pericaria in the sensory cortex; f very strong immunoreactivity of processes and motoneuron pericaria in control spinal cord anterior horn. Bars 100 μm each

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques: Immunohistochemistry, Control

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: Representative immunohistochemistry for dynactin DCTN3 on sections from SALS and control human cases. a Weakly immunopositive single neuron in SALS motor cortex ( arrow ) and visible loss of neurons; b mostly negative immune reaction in SALS neurons and axons ( arrow ) in the sensory cortex; c very poor or absent immunoexpression in the preserved motoneurons with features of degeneration in the anterior horn of SALS spinal cord; d weak immunolabel in pericaria of neurons and pronounced in their processes in the control motor cortex; e strong immunoreactivity of axons and poor of neuronal pericaria in the control sensory cortex; f weak immunoexpression in control pericaria of anterior horn motoneurons and mild in axons ( arrows ). Bars 100 μm

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques: Immunohistochemistry, Control, Immunolabeling

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: Expression of dynactin Dctn1 or Dctn3 mRNA in the CNS of transgenic mice. The expression was determined by RT-PCR and expressed as the ratio of the optical density (OD) value of Dctn1 or Dctn3 to the optical density of S12 protein RNA, as indicated in the Material and Method section. Filled diamond dashed lines wild-type controls (+/+); filled triangles Cra1/+ mice; filled circles SOD1/+ mice; filled squares Cra1/SOD1 hybrides

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques: Expressing, Transgenic Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: Percentage of dynactin subunits expression in the CNS of transgenic mice

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques: Expressing, Transgenic Assay