832/3 cells 48 hrs post siRNA transfections were seeded at low density on HTB9 coated-slides (Hayes et al., 2017 (link)) and cultured overnight. Following treatments, cells were fixed in 10 % neutral-buffered formalin and incubated overnight with antibodies raised against insulin (Dako), chromogranin A (Abcam), chromogranin B (Abcam), and TGN46 (Abcam) as indicated. AlexaFluor conjugated secondary antibodies (Invitrogen) were used for detection. Cells were counterstained with DAPI (Sigma) and mounted using Fluorosave (Calbiochem). Images were captured using a Zeiss Axiophot microscope and analyzed by Fiji software.
Tgn46
Manufactured by Abcam
Sourced in United Kingdom
TGN46 is a trans-Golgi network (TGN) membrane protein. It is commonly used as a marker for the trans-Golgi network in immunocytochemistry and Western blot applications.
Automatically generated - may contain errors
Lab products found in correlation
Gm130, by BD (3 mentions)
α tubulin, by Merck Group (2 mentions)
Injectman ni2, by Eppendorf (2 mentions)
Ergic 53 p58, by Merck Group (2 mentions)
X tremegene 9 transfection reagent, by Roche (2 mentions)
Sodium butyrate, by Merck Group (2 mentions)
Chromogranin a, by Abcam (1 mentions)
Axiophot microscope, by Zeiss (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Calreticulin, by Abcam (1 mentions)
Lamp1, by Cell Signaling Technology (1 mentions)
Ha tag, by Cell Signaling Technology (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit alexa 488, by Thermo Fisher Scientific (1 mentions)
P16ink4, by BD (1 mentions)
Nf κb p65, by Santa Cruz Biotechnology (1 mentions)
C ebpβ, by Santa Cruz Biotechnology (1 mentions)
Lamp2, by BD (1 mentions)
Goat anti rabbit alexa 546, by Thermo Fisher Scientific (1 mentions)
9 protocols using tgn46
1
Immunofluorescence Imaging of Endocrine Cells
2
Immunofluorescence Microscopy of Transfected Cells
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Cells were seeded over glass coverslip in 24-wells plates and plasmid transfected the day after with Lipofectamine-2000. The following day cells were fixed with fresh 4% PFA pH8.0, permeabilized and blocked with PBS containing 0.1% Triton X-100 and 10% heat-inactivated normal goat serum (PBSTH). Primary antibodies were incubated overnight at 4 °C at dilutions recommended by the providers in PBSTH. After washing in PBS, secondary fluorescently tagged antibodies were incubated for 1 h at room temperature in PBSTH. For non-permeabilized immunofluorescence with anti-HA antibodies the protocol of Briley et al. [100 (link)] was followed with the exception of the replacement of BSA for 10% heat-inactivated goat serum. The efficiencies of transfection and the total numbers of HA-positive cells in each experiment were determined per condition. Images were taken with an LSM700 confocal microscope (0.4 µm slices) at the University of Geneva’s Imaging Facility and processed using FIJI software. Primary antibodies used were anti-: HA-Tag (Cell Signaling 2367; 1/100), TGN46 (ABCAM ab50595, 1/200), cMYC (Santa Cruz Biotechnology sc-40; 1/100), CALRETICULIN (ABCAM ab2907;1/75), EEA1 (Cell Signaling No. 3288; 1/100), and LAMP1 (Cell Signaling No. 9091; 1/200).
3
Comprehensive Antibody Profiling for Cellular Analysis
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The antibodies used in this study were as follows: TGN46 (Abcam; ab16052); CCM3 (Acris; AP26023PU-N); BrdU (BD Biosciences; 555627), Lamp-2 (BD Biosciences; 555803), p21CIP1 (F-5) (Santa Cruz; sc-6246), p16INK4 (BD Biosciences; 511325), p62 (BD Biosciences; 610832); GAPDH (Calbiochem; CB-1001); LC3B (Cell Signaling; 3868), mTOR (Cell Signaling; 2983); H2AX (Ser139) (Millipore; 05-636); IL-8 (500-P28) (PeproTech; 500-P28); NFκB p65 (Santa Cruz; sc-8008); p53 (DO-1) (Santa Cruz; sc-126); C/EBPβ (Santa Cruz; sc-150); and tubulin (T5168) (Sigma-Aldrich). The secondary antibodies used were as follows: goat anti-rabbit DyLight™ 800, goat anti-mouse DyLight™ 680 (Thermo Scientific); goat anti-mouse Alexa 488, goat anti-rabbit Alexa 488, goat anti-mouse Alexa 594, and goat anti-rabbit Alexa 546 (Molecular Probes).
All plasmids were constructed using standard molecular biology techniques.
All plasmids were constructed using standard molecular biology techniques.
4
Visualizing Golgi Apparatus Dynamics
5
Immunohistochemical Analysis of Mouse Tissues
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Male mice were sacrificed and the tissues were immediately embedded in optimum cutting temperature compound (OCT, Tissue‐Tek) and cut into 8 μm sections using a microtome‐cryostat (LeicaCM1950, Wetzlar, Germany). Frozen sections were fixed with 4% paraformaldehyde for 20 min and washed in PBS three times. After being blocked with 5% BSA for 30 min, the sections were incubated with primary antibody (sp56, QED Bioscience, 55101; Lectin PNA, Alexa Fluor™ 488 Conjugate, Thermo Fisher Scientific, L21409; GM130, BD Bioscience, 610822; TGN46, Abcam, ab16059) at 4°C overnight. After being washed with PBS, the samples were incubated with secondary antibody, diluted with PBS for 1 h at 37°C, washed with PBS and stained with DAPI. The slides were then mounted and viewed. The images were acquired using an FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).
6
Retromer Detection Immunolabeling Protocol
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For retromer detection, antibodies against Vps26 (Abcam, Cambridge, UK), Vps35 (Novus Biologicals, Littleton, CO, USA) and sorLA (from Merck Millipore, St. Louis, MO, USA, for immunocytochemistry and BD, Franklin Lakes, NJ, USA, for Western blot analysis) were used. The following antibodies were used as organelle markers: TGN46 (Abcam, Cambridge, UK), EEA1 (Cell Signaling Technology, Danvers, MA, USA), Rab7 (Abcam, Cambridge, UK), transferrin receptor (Life Technologies, Waltham, MA, USA) and LAMP1 (Sigma, St. Louis, MO, USA and Santa Cruz, Dallas, TX, USA). For APP labelling N-terminal antibody (APP-NT, 22C11, Merck Millipore, St. Louis, MO, USA) and C-terminal antibody (APP-CT, Y188, Abcam, Cambridge, UK) were used. Antibodies against β-actin were purchased from Abcam (Cambridge, UK), TUJ-1 from Covance (Denver, PA, USA) and calbindin from Swant (Burgdorf, Switzerland).
7
Organelle Trafficking Protein Analysis
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The primary antibodies were: a) rabbit polyclonal – giantin, PDIA3, ASGP-R1 (Abcam); b) rabbit monoclonal – GM130, SAR1B (Abcam); c) mouse monoclonal – SAR1A, giantin and TGN46 (Abcam), β-actin (Sigma), GRASP65, Sec24d (Santa Cruz Biotechnology), giantin (Abcam); goat polyclonal – COPII (Sec23a, Abcam). The secondary antibodies (Jackson ImmunoResearch) were: a) HRP-conjugated donkey anti-rabbit, donkey anti-mouse, and donkey anti-goat for Western-blotting; b) donkey anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594. Pyrazole (Sigma), MG-132 (EMD Chemicals) and Brefeldin A (Sigma) were dissolved in dimethyl sulfoxide (DMSO) immediately before use. Cells treated with a corresponding concentration of DMSO served as controls. The regular working concentrations were: Pyrazole – 5 mM for 72 h, MG-132 – 2.5 μM for 24 h and Brefeldin A – 36 μM for 1 h.
8
Intracellular Trafficking in CHO and HeLa Cells
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CHO and HeLa cells were purchased from ATCC (Manassas, VA). DMEM, Advanced DMEM, DMEM/F12, Penicillin, Streptomycin, Geneticin, Lipofectamine 200 and Alexa546-conjugated transferrin were all obtained from Life Technologies (Gaithersburg, MD). pCI vectors were from Promega (Madison, WI). Fluorescent constructs were obtained from OriGene (Rockville, MD) and AddGene (Cambridge, MA). Antibodies used were LMTK2 (Rabbit monoclonal, Sigma Aldrich, St Louis MO; note the antibody recognizes a region distal to the putative di-acidic motif [22 (link), 29 (link)]), TGN46 (Rabbit Polyclonal, Abcam, Cambridge MA), EEA1 (Rabbit monoclonal, Cell Signaling, CA), PDI (Rabbit polyclonal, Santa Cruz, CA), GAPDH (rabbit polyclonal; Santa Cruz). Secondary antibodies were goat anti-rabbit IRDye 680RD conjugates (Licor, Lincoln, NE), and all images were collected on a Licor Odyssey SA imager. For immunofluorescence microscopy, labeled secondary antibodies (cy3 and alexa 488 conjugates) were from Jackson Laboratories (West Grove, PA). All other reagents were obtained from Sigma and were of reagent grade.
9
Visualizing Golgi Apparatus Dynamics
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HeLa cells were microinjected with EspG proteins using a semiautomatic InjectMan NI2 (Eppendorf) with a needle concentration of 25 μM unless stated otherwise. Transfections were performed using XtremeGene 9 Transfection Reagent (Roche) for 16–18 hr. Expression of NAGT I in NAGFP cells was stimulated by the addition of 5 μM sodium butyrate (Sigma-Aldrich) to the media. AMCA-EspG was produced using an NHS-AMCA labeling kit (Pierce). BFA and nocodazole treatments were performed at 5 μg ml−1 for 30 min and 30 μM for 2 hr, respectively. Cellular markers were detected using following antibodies: GM130 (BD Transduction Labs), ERGIC-53/p58 (Sigma-Aldrich), TGN46 (Abcam), β-COP (EAGE, Joachim Seeman, UTSW), and α-tubulin (Sigma-Aldrich).
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