VSMCs were lysed with sample buffer containing 0.1 μmol/L DTT and 0.1% of Triton X-100. For preparing cytosolic and nuclear fractions from cells, separate buffers were prepared. Samples were separated using SDS-PAGE and blotted onto polyvinylidene fluoride (PVDF) membranes. Analysis was done based on a chemiluminescence-based detection system (ImageQuant LAS 4000 mini). Primary antibodies used were rabbit anti-zyxin (B71, provided by M. Beckerle, Huntsman Cancer Institute, Salt Lake City, UT) (1:1000), goat anti-MRTF-A 1:1000 (Santa Cruz Biotechnology), rabbit anti-tubulin 1:1000 (Cell Signaling), mouse anti-RhoA 1:500 (Cytoskeleton), and rabbit anti-LPP 1:1000 (Atlas antibodies).
Rabbit anti tubulin
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Rabbit anti-tubulin is a primary antibody that recognizes the tubulin protein, a key component of the cytoskeleton. It can be used to detect and locate tubulin in biological samples using techniques such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti gapdh, by Cell Signaling Technology (5 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Horseradish peroxidase linked anti rabbit igg, by Jackson ImmunoResearch (3 mentions)
Rabbit anti β actin, by Cell Signaling Technology (3 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (3 mentions)
Rabbit anti histone h3, by Cell Signaling Technology (3 mentions)
Rabbit anti actin, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Rabbit anti parp, by Cell Signaling Technology (2 mentions)
Rabbit anti sting, by Cell Signaling Technology (2 mentions)
Mouse anti tmprss2, by Santa Cruz Biotechnology (2 mentions)
Odessy scanner, by LI COR (2 mentions)
Goat anti rabbit igg dylight 800, by Thermo Fisher Scientific (2 mentions)
Goat anti ace2, by R&D Systems (2 mentions)
Rabbit anti irf1, by Cell Signaling Technology (2 mentions)
Goat anti mouse igg dylight 680, by Thermo Fisher Scientific (2 mentions)
Mouse anti cathepsin l, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti mavs, by Thermo Fisher Scientific (2 mentions)
Mouse anti rig 1, by Adipogen (2 mentions)
Rabbit anti mda 5, by Proteintech (2 mentions)
30 protocols using rabbit anti tubulin
1
Subcellular Protein Fractionation and Analysis
2
Western Blot Analysis of Cell Lysates
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Cell lysates were prepared in 1 × RIPA Lysis buffer (Millipore) supplemented with 1:100 Protease Inhibitor Cocktail (Sigma), 1:100 Phosphatase Inhibitor Cocktail 2(Sigma) and 1:100 Phosphatase Inhibitor Cocktail 3 (EMD Chemical/Calbiochem). In total, 10 μg of protein from each sample was separated using SDS–polyacrylamide gel electrophoresis, transferred to a PVDF membrane and blocked with 5% milk (Biorad). Primary antibodies used were 1:1,000 Mouse anti-E-cadherin (BD Biosciences, BDB610181), 1:500 mouse anti-Vimentin (Sigma, V5255) and 1:1,000 rabbit anti-Tubulin (Cell Signaling, 21485). HRP-conjugated anti-mouse (1:10,000) and anti-rabbit (1:10,000) secondary antibodies (Biorad) were allowed to bind before washing and probing the bands using Luminata Classico Western HRP substrate (Millipore). Unedited scans of the original blots are presented in Supplementary Fig. 7 .
3
Western Blot Protocol for Stem Cell Markers
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Total protein was extracted using RSB100 buffer as previously described24 (link). Electrophoresis of denatured samples was conducted with Tris-glycine buffered sodium dodecyl sulfate polyacrylamide gel electrophoresis at 130 V for 1.5 h and proteins were then transferred at 90 V for 1 h by wet transfer to a nitrocellulose membrane. The membrane was blocked with 5% milk in tris-buffered saline (TBS) at room temperature for 1 h, then primary antibodies mouse anti-EOMES (clone 644730/catalog # MAB6166, R&D Systems), goat anti-SOX17 (catalog # AF1924, R&D Systems), rabbit anti-NKX2.5, rabbit anti-AFP (catalog # SAB3500533, Sigma-Aldrich), and rabbit anti-Tubulin (catalog # 2148 S, Cell Signaling Technologies) were used to probe for the specified antigen overnight at 4°C. Membranes were washed three times in TBS with 0.1% Tween-20 (TBST) and probed with infrared dye–conjugated secondary antibodies, all from LI-COR (Lincoln, NE, USA): IRDye 800CW donkey anti-rabbit IgG (1:15,000 dilution; catalog # 926-32213), IRDye 680RD donkey anti-goat IgG (1:20,000 dilution; catalog # 926-68074), or IRDye 680RD donkey anti-mouse IgG (1:20,000 dilution; catalog # 926-68072), then washed three additional times with TBST. Proteins were visualized by scanning on the Odyssey CLx system (LI-COR).
4
Western Blot Analysis of Apoptotic Markers
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At the end of the incubation periods, islets and INS-1E cells were washed in ice-cold PBS and lysed in RIPA lysis buffer containing 50 mM Tris HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS supplemented with Protease- and phosphatase inhibitors (Pierce, Rockford, IL, USA). Protein concentrations were determined with the BCA protein assay (Pierce). Equivalent amounts of protein from each treatment group were run on a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and electrically transferred onto PVDF membranes. After blocking by 2.5% milk (Cell Signaling) and 2.5% BSA, membranes were incubated overnight at 4 °C with rabbit anti-cleaved caspase-3 (#9664), rabbit anti-PARP (#9532), rabbit anti-cleaved PARP (rat specific #9545), rabbit anti-phospho YAP(S127) (#4911), rabbit anti-LATS2 (#5888), rabbit anti-tubulin (#2146), rabbit anti-GAPDH (#2118), rabbit anti-β-actin (#4967) (all Cell Signaling Technology), and rabbit anti-PDX1 (#47267) and rabbit anti-p-MST1 (#79199) (both from Abcam) antibodies, all at a dilution of 1:1000, followed by horseradish-peroxidase-linked anti-rabbit IgG (Jackson). Membrane was developed by using a chemiluminescence assay system (Pierce) and analyzed using DocIT®LS image acquisition 6.6a (UVP BioImaging Systems, Upland, CA, USA). Uncropped and unprocessed scans of all Western blots are available in the Source Data file.
5
Fly Head Protein Extraction and Western Blot
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Fly heads were homogenized on ice in radioimmune precipitation buffer (RIPA, containing 50 mm Tris–HCl, 150 mm NaCl, 0.1% sodium deoxycholate [SDS],, 0.5% Na, 1% NP-40, [pH 8.0]) supplemented with protease inhibitor (Complete Protease inhibitor, Roche, Indianapolis, IN, USA). Homogenates were incubated on ice for 20 min and protein lysates were extracted by centrifugation (12000 rpm for 20 min at 4°C). Supernatants were used for immunoblotting. Protein concentrations were measured by Micro BCA assay kit (ThermoFisher Scientific; 23235). Western blotting was performed as described before (25 (link)). Briefly, 30 μg of protein was loaded on a 7.5% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. Membranes were blocked in 5% skimmed milk in PBS and probed with primary antibodies. Immunodetection was performed with specific secondary antibodies conjugated to horseradish peroxidase and the ECL-plus chemiluminescent detection system (Amersham Biosciences, Little Chalfont, UK) with bands quantified on a LAS-3000 Station (GE Healthcare, Chicago, IL, USA). The signal was normalized to the signal obtained from tubulin for quantification. Primary antibodies were used in a 1/500 dilution: rabbit anti-FUS (Bethyl Laboratories, A300-302A) and rabbit anti-tubulin (Cell signaling, #2125).
6
Western Blot Analysis of Key Proteins
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Cells washed with phosphate-buffered saline (PBS) were lysed in SDS buffer and boiled at 94 °C for 5 min. After measuring protein quantity by Bradford, equal amounts of protein were resolved by SDS-PAGE, transferred to a nitrocellulose membrane (Millipore) and probed with one of the following antibodies: mouse anti-Chk1 (1:1000, abcam), rabbit anti-p21 (1:500, Cell signaling), rabbit anti- p53 (1:1000, Cell Signaling), rabbit anti-SETD8 (1:1000, Cell Signaling), mouse anti-β-actin (1:20,000, Sigma), rabbit anti-H2A.X and anti-phospho-H2A.X-Ser139 (1:1000, Cell signaling), rabbit anti-H4-K20me1 (1:1000 Cell Signaling), and rabbit anti-Histone H4 (1:1000, Cell Signaling), rabbit anti-tubulin (1:1000, Cell signaling). Membranes were then incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. The immunoreactive bands were detected by chemiluminescence (Pierce).
7
Extraction and Analysis of Cellular Proteins
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Cytoplasmic and nuclear extracts from RL95-2 cells were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce; Thermo Scientific, Rockford, IL, USA). Primary antibodies were the following: rabbit anti-Pak4 (1:1000, Abcam, Cambridge, UK), rabbit anti-p-Pak4 Ser474 (1:1000, Cell Signaling Technology), mouse anti-β-actin (1:2000, ProteinTech Group, Chicago, IL), rabbit anti-tubulin (1:1000, Cell Signaling Technology), and rabbit anti-histone H3 (1:1000, Cell Signaling Technology).
8
Western Blot Analysis of Signaling Proteins
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Cells were lysed in Laemmli sample buffer and run in 4–20% Mini-PROTEAN TGX precast protein gels (Bio-Rad Laboratories). The primary antibodies used were rabbit anti-Egr1 (no. 4154; Cell Signaling), rabbit anti–c-Fos (no. 2250; Cell Signaling), rabbit anti–c-Jun (no. 9165; Cell Signaling), mouse anti-Snail (no. 3895; Cell Signaling), rabbit anti-PARP1 (no. 9532; Cell Signaling), rabbit anti-Smad2/3 (no. 8685; Cell Signaling), rabbit anti-pSmad2/3 (no. 8828; Cell Signaling), mouse anti–cytochrome c (no. sc-13560; Santa Cruz Biotechnology), mouse anti-Cox4 (no. 11967; Cell Signaling), rabbit anti–caspase 9 (no. 9502; Cell Signaling), mouse anti–caspase 8 (no. 9746; Cell Signaling), rabbit anti-Tubulin (no. 2128; Cell Signaling), and mouse anti–α-Tubulin (T6199; Sigma). The secondary antibodies used were IRDye 800CW donkey anti–rabbit IgG (H+L), IRDye 680LT donkey anti–mouse IgG (H+L), and IRDye 800CW donkey anti–mouse IgG (H+L; LI-COR Biosciences). The blots were scanned on an Odyssey imaging system (LI-COR Biosciences). Cell treatment, sample collection, and Western blotting were repeated at least three times, and the representative blots are shown in the figures.
9
Quantitative Western Blot Analysis of Focal Adhesion Kinase
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Cell extracts were prepared by adding lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.5% Nonidet-40, 1 mM dithiothreitol) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Cells were removed with a cell scraper, incubated with lysis buffer at 4 °C for 2 h and then centrifuged at 16100 × g. Supernatants were collected and treated with SDS sample buffer. Western blot analyses were performed with rabbit anti-FAK (1:1000, Cell Signaling) and rabbit anti-tubulin (1:2000, Cell Signaling) antibodies. The peroxidase-conjugated anti-rabbit IgG was used as secondary antibody. ImageJ software (https://imagej.nih.gov/ij/ ) was used for the quantitative analysis and different gel images were quantified using linear signal ranges.
10
Characterization of FLT3 Receptor Mutants
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Human recombinant FLT3 ligand was from ORF genetics (Kópavogur, Iceland). The transfection reagent Lipofectamine 2000 was from Thermo Scientific and cycloheximide was from Sigma-Aldrich. pcDNA3-FLT3-WT, pMSCVpuro-FLT3-WT and pMSCVpuro-FLT3-ITD were described previously [18 (link)]. pMSCVpuro-FLT3-WT/Y842F and pMSCVpuro-FLT3-ITD/Y842F plasmids were generated by site-directed mutagenesis using QuikChange mutagenesis XL kit (Agilent Technologies). The anti-FLT3 antibody was a rabbit polyclonal antibody produced in-house. Mouse monoclonal anti-beta-actin, horseradish peroxidase-conjugated anti-FLAG antibody and mouse monoclonal anti-FLAG antibodies were from Sigma-Aldrich. Mouse anti-phosphotyrosine (4G10) antibody and mouse mono-ubiquitin antibody were from Millipore and Covance Research Products, respectively. Rabbit anti-ERK2, rabbit anti-phospho ERK1/2 (pThr202/pThr204), goat anti-AKT antibodies were from Santa Cruz Biotechnology. Rabbit anti-tubulin, rabbit anti-phospho-AKT (pSer473) rabbit anti-phospho GAB2 and rabbit anti-phospho-SHP2 were from Cell Signaling Technology.
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