Journal: Drug Design, Development and Therapy

Article Title: Modes of cell death induced by tetrahydroisoquinoline-based analogs in MDA-MB-231 breast and A549 lung cancer cell lines

doi: 10.2147/DDDT.S152718

Figure Lengend Snippet: Immunofluorescent images indicated a disrupted microtubule morphology in compound-exposed A549 ( Ai – Gi ) and MDA-MB-231 ( Aii – Gii ) cells. Notes: Microtubules (α-tubulin) are stained in green and DNA is stained in blue with DAPI. Intact microtubule networks were visualized in DMSO-exposed cells ( Ai , Aii ). Colchicine-treated cells ( Bi , Bii ) were used as a positive control for microtubule disruption and depolymerization. Cells exposed to 2-ME ( Ci , Cii ), STX 2895 ( Di , Dii ), STX 3329 ( Ei , Eii ), STX 3450 ( Fi , Fii ) and STX 3451 ( Gi , Gii ) showed a decrease in cell density, DNA fragmentation and microtubule disruption (80× magnification, scale bar: 10 µM). Abbreviations: 2-ME, 2-methoxyestradiol; DMSO, dimethyl sulfoxide; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Cells were incubated in the primary antibody cocktail containing the primary monoclonal antibody to tubulin alpha (clone DM1A) purchased from Imgenex (San Diego, CA, USA; 1:4,000 ratio) in PBS containing 0.3% bovine serum albumin (BSA; 30 min at RT).

Techniques: Staining, Positive Control, Disruption

Journal: Scientific Reports

Article Title: Disrupted-in-schizophrenia-1 (DISC1) Regulates Endoplasmic Reticulum Calcium Dynamics

doi: 10.1038/srep08694

Figure Lengend Snippet: (a) The subcellular fractionation procedure using sucrose gradients ( 1 ) and subcellular localization of DISC1 ( 2i ) or DISC1 fragments ( 3i ) in HEK293 cells. Calnexin, mitofilin and α-tubulin were used as ER, mitochondrial and cytosolic markers, respectively. (b) Co-localization between Flag-DISC1 and GFP-ER in HEK293 cells ( i ), Flag-mDISC1 and GFP-ER in mouse hippocampal neurons at DIV10 ( ii ). Scale bars represent 10 μm. (c) Electron micrograph with pre-embedding immunogold staining. Ultrastructure of Flag-DISC1 in HEK293 cells showing the existence of DISC1 in the ER (red arrowhead) and mitochondria (black arrowheads) ( i ). Double immunogold labeling of Flag-DISC1 with calnexin in HEK293 cells ( ii ) and endogenous mDISC1 with calnexin in adult mouse brain sections ( iii ). Immunogold particles (6 nm) for Flag-DISC1 and endogenous mDISC1 are indicated with black arrows and 15 nm immunogold particles for calnexin with red arrows. The magnified images are represented with bold lines and scale bars represent 200 nm ( i ) and 100 nm or 50 nm ( ii , iii ). Western blotting was performed under the same experimental conditions in each panel and full-length blots are presented in .

Article Snippet: Rabbit anti-mitofilin (NB100-1919), rabbit anti-calnexin (SPA-865) and mouse anti-α-tubulin (DM1A) antibodies were purchased from NOVUS Biologicals, Assay Designs and Santa Cruz Biotechnology, respectively.

Techniques: Fractionation, Staining, Labeling, Western Blot

Journal: European Journal of Histochemistry : EJH

Article Title: Immunohistochemical Demonstration of Specific Antigens in the Human Brain Fixed in Zinc-ethanol-Formaldehyde

doi: 10.4081/ejh.2015.2530

Figure Lengend Snippet: Characteristics of the antibodies used and details of their application

Article Snippet: α-Tubulin , Mouse monoclonal (clone DM-1A); BioGenex, Fremont, CA, USA; MU121-UC; 1:100 , 60 min, 40°C , MACH2.

Techniques: Incubation

Journal: European Journal of Histochemistry : EJH

Article Title: Immunohistochemical Demonstration of Specific Antigens in the Human Brain Fixed in Zinc-ethanol-Formaldehyde

doi: 10.4081/ejh.2015.2530

Figure Lengend Snippet: Examples of immunohistochemical staining of the human brain fixed in the ZEF. A) Substantia nigra, pars reticulata : α-tubulin in the single neuron with extensively stained perikaryon and processes, many immunoreactive fibers in the surrounding neuropil. B) Nucleus ruber: extensive neuron-specific enolase (NSE) immunoreactivity in the nucleus, perinuclear cytoplasm, and processes of neurons; some bright NSE-immunoreactive fibers and medium-intensity staining of the neuropil are also seen. C) Caudate nucleus (head): choline acetyltransferase (ChAT) immunoreactivity in the multipolar neuron and its processes. D) Thoracic sympathetic ganglion: tyrosine hydroxylase (rabbit polyclonal) immunoreactivity in most of large and small neurons, mainly in the cytoplasm, as well as in bundles of nerve fibers going alongside these neurons. E) Substantia nigra, pars compacta : tyrosine hydroxylase (mouse monoclonal) antibody labels many (but not all) nigral neurons. F) Nucleus ruber: NeuN-labeled neurons with highly stained nucleus and the perinuclear cytoplasm. G) Parietal cortex, layer III: Iba-1 distinctly labels multiple thin processes and some perikarya of microgliocytes. H) Cerebellum: strong calbindin immunostaining in the cytoplasm and dendrites of the Purkinje cells. I) Cerebellum: calretinin immunohistochemistry visualizes the unipolar brush cell, a rare sort of the large cerebellar interneurons in the granular layer. J) S ubstantia nigra, pars reticulata : distinct synaptophysin immunoreactivity in the clusters of synapses alongside the neuronal and fiber plasmalemma. K) Cerebellar cortex: GAD65 immunostaining of the fine fiber nets in the granular layer, which localization coincides with the distribution of the glomeruli, the specific synaptic formations composed by the granular cell dendrites, mossy fibers, and axons of the Golgi cells. L) Nucleus ruber: GFAP distinctly labels the perinuclear cytoplasm and processes of fibrous astrocytes forming large nets in the neuropil. Preparations A-E), G-L) are counterstained with hematoxylin; F) with astra blue.

Article Snippet: α-Tubulin , Mouse monoclonal (clone DM-1A); BioGenex, Fremont, CA, USA; MU121-UC; 1:100 , 60 min, 40°C , MACH2.

Techniques: Immunohistochemical staining, Staining, Labeling, Immunostaining, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: The PX-RICS-14-3-3?/? Complex Couples N-cadherin-?-Catenin with Dynein-Dynactin to Mediate Its Export from the Endoplasmic Reticulum *

doi: 10.1074/jbc.M109.081315

Figure Lengend Snippet: 14-3-3ζ/θ is involved in ER-to-Golgi transport of the N-cadherin-β-catenin complex. A, knockdown of 14-3-3ζ or -θ results in the disappearance of N-cadherin and β-catenin at the cell-cell boundaries and the ER accumulation of N-cadherin. HeLa cells transfected with the indicated siRNAs were subjected to immunofluorescent staining with anti-N-cadherin plus anti-calnexin or anti-β-catenin antibody. Arrows indicate N-cadherin or β-catenin enriched at the sites of cell-cell contact. Arrowheads indicate the absence of N-cadherin and β-catenin at the borders between two neighboring cells. Scale bars, 10 μm. B, upper panel, knockdown of 14-3-3ζ or -θ inhibits the surface expression of N-cadherin. The amounts of total and surface N-cadherin in siRNA-transfected HeLa cells were evaluated by surface biotinylation assays. Transferrin receptor (TfR) and α-tubulin were used for positive and negative controls, respectively. Lower panel, surface expression of N-cadherin was quantified by measuring the band intensity of the biotinylated fraction (SA pulldown in the upper panel) compared with that of total input. Representative results of the three independent experiments are shown. C, Ca2+-dependent cell adhesion is abrogated by knockdown of 14-3-3ζ or -θ. HeLa cells expressing the indicated siRNAs were dissociated by pipetting in the presence of Ca2+. Cell adhesion activity was quantified by counting the total number of cells (Nc) and the number of particles (cell clumps) (Np). Error bars represent the mean ± S.D. (n = 6).

Article Snippet: Mouse anti-14-3-3ϵ, mouse anti-β-catenin, and mouse anti-p150 Glued antibodies were purchased from BD Biosciences; mouse anti-GFP antibody (Living colors A.v. monoclonal antibody (JL-8)) was from Invitrogen; rabbit anti-Myc antibody was from MBL; mouse anti-α-tubulin antibody (DM1A) was from Merck; mouse anti-N-cadherin (13A9) and mouse anti-DYNC1I (74.1) antibodies were from Millipore; mouse anti-Myc (9E10), mouse anti-pan-14-3-3 (H-8), rabbit anti-14-3-3ζ (C-16), goat anti-14-3-3β (A-15), goat anti-14-3-3γ (A-12), goat anti-14-3-3η (E-12), and goat anti-Sec23 (E-19) antibodies were from Santa Cruz Biotechnology; mouse anti-FLAG (M2) and mouse anti-14-3-3θ (3B9) antibodies were from Sigma; rabbit anti-calnexin antibody was from Stressgen; and mouse anti-transferrin receptor antibody (H68.4) was from Zymed Laboratories Inc.. Rabbit and mouse IgGs were from Millipore.

Techniques: Transfection, Staining, Expressing, Activity Assay