Particle uptake was confirmed using laser scanning microscopy (LSM) to detect Atto647 emission in cells. PHNECs were fixed in 70% ethanol and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich). Cells were stained with primary antibodies mouse anti-β-tubulin (1:100; Life Technologies), rabbit anti-occludin (1:50; Molecular Probes) or rabbit anti-mucin (1:50; Santa Cruz Biotechnology) overnight at 4°C. The following secondary antibodies were used for 1h at room temperature: goat anti-mouse Alexa488 (1:200; Molecular Probes), goat anti-rabbit Alexa546 (1:200; Life Technologies) and phalloidin Atto390 (1:25; ATTO-TEC). Cells were washed and embedded in Aquatex mounting medium (Merck KGaA, Darmstadt, Germany). Optical sections were taken with a Zeiss LSM 710 (Carl Zeiss AG, Feldbach, Switzerland) with a 40x oil objective (Plan-Apochromat 40x/1.40 Oil) and a digital zoom of 1.4x. Image processing was performed using Imaris (Bitplane AG, Zurich, Switzerland) software.
Mouse anti β tubulin
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Mouse anti-β-tubulin is a primary antibody that specifically binds to the beta-tubulin protein. Beta-tubulin is a major component of the cytoskeleton and is involved in the formation of microtubules. This antibody can be used to detect and visualize beta-tubulin in various cell and tissue samples using techniques such as immunofluorescence, Western blotting, and immunohistochemistry.
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Lab products found in correlation
Rabbit anti lamin b1, by Abcam (2 mentions)
Pathscan multiplex western cocktail 1, by Cell Signaling Technology (2 mentions)
Phospho akt t308, by New England Biolabs (2 mentions)
Phospho p44 42 mapk erk1 2 t202 y204, by New England Biolabs (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Goat anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Aquatex mounting medium, by Merck Group (1 mentions)
Goat anti rabbit alexa 546, by Thermo Fisher Scientific (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Imaris, by Oxford Instruments (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Goat anti mouse alexafluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti β tubulin, by Thermo Fisher Scientific (1 mentions)
Ecl chemiluminescence kit, by GE Healthcare (1 mentions)
Mouse monoclonal anti ha clone 16b12, by Fortrea (1 mentions)
Mouse anti gapdh, by Thermo Fisher Scientific (1 mentions)
Mouse anti ub, by Santa Cruz Biotechnology (1 mentions)
Mouse anti flag, by Proteintech (1 mentions)
Mouse anti gfp, by Abmart (1 mentions)
Rabbit anti sumo1, by Abcam (1 mentions)
11 protocols using mouse anti β tubulin
1
Microscopic Analysis of Particle Uptake in Cells
2
Measuring DNA Damage Response to PARP Inhibitors
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Cells were seeded as for PARP-1 activity assay. Fresh medium was added containing rucaparib or olaparib, and cells were simultaneously irradiated, before incubating for 1.5 h at 37 °C. After treatment, cells were fixed with 4 % (w/v) paraformaldehyde for 30 min at room temperature before blocking with 2 % (w/v) BSA (in PBS) for 30 min at room temperature. Fixed cells were then incubated overnight at 4 °C with a 1:50 dilution of rabbit anti-phospho-Histone H2AX(Ser139)-Alexa Fluor 647-conjugated monoclonal antibody (Cell Signalling Technology, supplied by New England Biolabs, Hitchin, UK, Cat# 9720), followed by overnight incubation with a 1:250 dilution of mouse anti-β-tubulin (Life Technologies, Paisley, UK) in antibody buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1 % (w/v) BSA in distilled water). Bound anti-β-tubulin primary antibody was visualised after 1 h incubation at room temperature using goat anti-mouse Alexa Fluor 488-conjugated secondary antibody (Life Technologies, Paisley, UK; Cat# A11029), at a dilution of 1:500 in antibody buffer. Cells were mounted and fluorescence visualised as for PARP-1 activity assay.
3
Preparation and Separation of Parasite Proteins
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For preparation of total protein parasite extracts, 1 × 10 7 epimastigotes were harvested by centrifugation at 1000 × g and disrupted with the addition of 5 × SDS-PAGE loading buffer (5 × SB). Clarified lysates and insoluble fractions from parasites were prepared as follows: 1 × 10 7 pelleted epimastigotes were resuspended in icecold PBS, disrupted by sonication (four pulses of 20 s each) and centrifuged at 20,000 ×g for 20 min. The fractions obtained (cytoplasmic supernatant and membranous pellet) were mixed separately with 5× SB in order to reach the same final volume for each sample. The different protein extracts were loaded onto 12% SDS-PAGE polyacrylamide gels and subjected to electrophoresis. Gels were transferred to nitrocellulose membranes and proteins detected using mouse monoclonal anti-HA (clone 16B12, Covance), mouse anti-β tubulin (Life Technologies), rabbit polyclonal anti-GFP or rabbit polyclonal anti-TcCyp19 antibodies (the last two kindly provided by Dr. Jaqueline Búa, ANLIS/Malbrán Institutes, Argentina). Horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgGs (Calbiochem) were used as secondary antibodies. All antibodies were used at 1:1000 dilutions in 3% bovine serum albumin in PBS (BSA-PBS) and detected with ECL™ chemiluminescence kit (GE Healthcare), according to the manufacturer's instructions.
4
Western Blot Analysis of Subcellular Proteins
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For Western blot analysis, cells were harvested and lysed in RIPA lysis buffer (Cell Signal Technology). To investigate subcellular distribution of proteins, nuclear and cytoplasmic fractions were enriched using Nuclear and Cytoplasmic Protein Extraction Kit (CoWin Bioscience). Western blot assays were followed according to an established protocol (20 (link)). Antibody used included mouse anti-GFP (Abmart), rabbit anti-acetyl Lysine (Abcam), rabbit anti-SUMO1 (Abcam), rabbit anti-Lamin B1 (Abcam), mouse anti-Ub (Santa Cruz), rabbit anti-USP49 (NOVUS), mouse anti-Flag (Proteintech), mouse anti-GAPDH (Thermo Fisher Scientific), mouse anti–β-tubulin (Thermo Fisher Scientific), rabbit anti-GRα (Abcam), rabbit anti-GRβ (Abcam). For protein stability detections, cells were treated with proteasome inhibitor MG132 (20 μmol/L, Sigma-Aldrich) for 6 hours or protein synthesis inhibitor Cycloheximide (CHX, 100 μg/mL, Sigma-Aldrich) for indicate time before harvest.
5
Comprehensive Antibody Panel Validation
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The following antibodies were used: mouse anti-GAPDH [6C5], rabbit anti-lamin B1, rabbit anti-cytochrome c [EPR1327], and rabbit anti- carbamoyl phosphate synthetase-1 (CPS1) [EPR7493] (Abcam, Cambridge, UK); mouse anti-β-tubulin; mouse anti-pan actin, mouse anti-hsp60 clone LK2 and mouse anti-Bax (Thermo Scientific, Waltham, MA); and rabbit anti-SAPK/JNK (Cell Signaling, Danvers, MA).
6
Insulin Signaling Pathway Activation
7
Insulin Signaling Pathway Activation
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Activation of the hIR was assessed by immunoblotting as previously described54 (link). L6 myoblasts overexpressing IR-A were stimulated with 10 nM insulin or mini-Ins for different times. Cell lysates were subjected to reducing 10 % SDS-PAGE, transferred to nitrocellulose and immunoblotted with the PathScan® Multiplex Western Cocktail I (Cell Signaling Technology #5301) against pAkt (Ser473), pERK1/2 (Thr202, Tyr204) and mouse anti-β-tubulin (Invitrogen #32–2600). Total Akt and ERK1/2 levels do not change over the time course measured (not shown). The β-tubulin was used as a loading control and pAkt and pERK1/2 were normalized against this control. Quantification of the blots was achieved using the Image Studio Lite quantification software. Activation was expressed as a percentage of the response to insulin at 10 min.
8
Neuronal Cytoskeletal and Synaptic Protein Analysis
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Immunostaining was performed to examine morphological changes in the neuronal cytoskeletal and synaptic proteins of cortical neurons cultured for 3 days and 10 days, respectively. Neurons were fixed with 4% paraformaldehyde and 15% picric acid for 1 h at room temperature. Permeabilization of fixed cultures was performed by incubation for 1 h in incubation media containing 5% goat serum and 0.1% Triton X. Cells were then incubated with primary antibodies rabbit anti-synaptophysin (1:500, Abcam) and mouse anti-PSD95 (1:2,000; NeuroMab) to study the development of synaptic proteins. Primary antibodies were applied at 4°C overnight. Following 3× wash with 1× PBS, secondary antibodies AlexaFluor 488 goat anti-rabbit IgG (1:200; Invitrogen) and AlexaFluor 546 goat anti-mouse IgG (1:200; Invitrogen) were applied for 1 h at room temperature. Mouse anti-β-tubulin (1:500; Invitrogen) was used to study the development of cytoskeletal proteins. Preparations were washed 3× in 1× PBS and mounted with MOWIOL mounting media. A Zeiss confocal microscope (LSM 510 Meta, Zeiss, Germany) was used to take fluorescence images. Image acquisition parameters such as exposure times, gain settings, laser intensity, pinhole size, etc., remained the same between control and treated cultures.
9
Fluorescent Visualization of Cytoskeletal Dynamics
10
Insulin Signaling Pathway Activation Assay
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Induction of signaling was assessed by immunoblotting as described51 (link). L6 myoblasts overexpressing IR-A (240,000 cells/well) were seeded in 6-well plates, grown to confluence (~48 hours), and stimulated with 10 nM human insulin or Vh-Ins-HALQ for different times. Lysates were precipitated with trichloroacetic acid, pH neutralized with 1M Tris pH8.0, separated on 10% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with primary antibodies for 16 h at 4 °C. Antibodies used were phospho-AKT (T308) (New England Biolabs #9275S), phospho p44/42 MAPK (ERK1/2) (T202/Y204) (New England Biolabs #9101S) and mouse anti-β-tubulin (Invitrogen #32–2600). β-tubulin was used as loading control for pAKT and pERK1/2 normalization. Quantitation of blots was performed using Image Studio Lite software. Activation was expressed as a percentage of the response to insulin at 10 min (three independent experiments).
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