Western blot analysis was performed as described previously with minor modification.16 (link),28 (link) For cPLA2 expression, primary antibodies included mouse monoclonal anti-cPLA2 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal rabbit anti–phospho(p)-cPLA2 antibody (1:500), and mouse anti–β-tubulin antibody (1:1,000; Sigma). For active caspase-3 and poly(adenosine diphosphate ribose) polymerase (PARP) expression, primary antibodies included rabbit anti–caspase-3 antibody (cleaved, 1:1,000), rabbit anti–PARP-1 (cleaved, 1:500), and mouse anti–β-tubulin antibody (1:1,000; Sigma). For extracellular signal-regulated kinase (ERK) expression, primary antibodies included polyclonal rabbit anti-ERK1/2 antibody (1:1,000) and monoclonal mouse anti–p-ERK1/2 antibody (1:2,000). Secondary Alexa Fluor 680 goat antimouse (1:10,000; Invitrogen, Grand Island, NY) and IRDye 800 goat antirabbit (1:5,000; Rockland, Gilbertsville, PA) antibodies were used. The Western blot was imaged and quantified using a Li-Cor Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln, NE) according to the manufacturer's instruction.
Mouse anti β tubulin antibody
Manufactured by Merck Group
Sourced in United States
The Mouse anti-β-tubulin antibody is a laboratory reagent used to detect the presence and distribution of the β-tubulin protein in cells and tissues. It is a primary antibody that specifically binds to the β-tubulin isoform, which is a key component of the cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Ecl prime western blotting detection reagent, by Cytiva (3 mentions)
Dc protein assay, by Bio-Rad (3 mentions)
Fusion fx imager, by Vilber (3 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Nitrocellulose membrane, by Abcam (2 mentions)
Alexa fluor 680 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Irdye 800 goat antirabbit, by Rockland Immunochemicals (1 mentions)
Prolong antifade kit, by Thermo Fisher Scientific (1 mentions)
Tcs sp laser scanning confocal microscope, by Leica (1 mentions)
Mouse anti tyrosine hydroxylase antibody, by Merck Group (1 mentions)
Rabbit anti flag monoclonal antibody, by Merck Group (1 mentions)
Triton x 100, by Avantor (1 mentions)
Mouse monoclonal anti flag antibody, by Merck Group (1 mentions)
Sarkosyl, by Avantor (1 mentions)
Ck 666, by Merck Group (1 mentions)
Alexa fluor 546 conjugate, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugate of phalloidin, by Thermo Fisher Scientific (1 mentions)
Revolve epi fluorescence microscope, by Echo Medical Systems (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
19 protocols using mouse anti β tubulin antibody
1
Quantitative Western Blot Analysis
2
Western Blotting of Hedgehog Pathway Proteins
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Western blots were carried out as previously described [13 (link)]. Total RIPA extracts from cells were prepared and protein concentration was estimated using the DC Protein Assay (Bio-Rad, Feldkirchen, Germany). An amount of 50 to 80 µg of proteins was separated on SDS-PAGE and transferred to membranes of nitrocellulose (Amersham, Bath, UK). After 1 h blocking in 5% non-fat milk, membranes were incubated overnight at 4 °C with rabbit anti-hPtch1 antibody (Abcam ab53715; 1/1000) or mouse anti-β-tubulin antibody (Sigma Aldrich, Merck, Darmstadt, Germany; 1/1000). After washing, membranes were incubated for 45 min with anti-rabbit (1:2000) or anti-mouse (1:5000) immunoglobulin coupled to horseradish peroxidase (Dako-Agilent, Santa Clara, CA, USA). ECL Prime Western Blotting detection reagent (Amersham) on a Fusion FX imager (Vilber Lourmat, Collegien, France) was used for detection.
3
Immunofluorescent Staining of Rat Striatal Neurons
4
Immunofluorescence Staining of Proteins
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Three dyes, ThT, ThS, and DAPI, and four antibodies, mouse anti-FLAG monoclonal antibody, rabbit anti-FLAG monoclonal antibody, 10 nm gold-labeled anti-mouse antibody, and mouse anti-β-tubulin antibody, were bought from Sigma-Aldrich (St. Louis, MO). Sarkosyl and Triton X-100 were purchased from Amresco (Solon, OH). Rabbit anti-SOD1 polyclonal antibody, mouse anti-SOD1 monoclonal antibody, and 10 nm gold-labeled anti-rabbit antibody were from Abcam (Cambridge, MA). Streptavidin, DyLight-405 conjugated secondary antibody and secondary antibody conjugated with Alexa 546/488 were from Invitrogen (Carlsbad, CA). Anti-dimedone polyclonal antibody was purchased from Millipore (Billerica, MA). Other chemicals were analytical grade, made in China.
5
Investigating Retinoic Acid and Arp2/3 Regulation
6
Patched Protein Expression in Melanoma
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Total RIPA extracts were prepared from melanoma cells grown to 80% confluence in 6-well plates. Protein concentrations were determined by the DC Protein Assay (Bio-Rad, Hercules, CA, USA). Samples (50 to 80 µg) were separated on SDS-PAGE and transferred to nitrocellulose membranes (Amersham, Courtaboeuf, France) using standard techniques. After 1 h at room temperature in blocking buffer (5% non-fat milk in PBS containing 0.1% Tween-20), nitrocellulose membranes were incubated overnight at 4 °C with rabbit anti-Patched antibody (Abcam ab53715; 1/1000, Cambridge, UK) or mouse anti-β-tubulin antibody (Sigma; 1/1000). After 3 washes, membranes were incubated for 45 min with anti-rabbit (1:2000) or anti-mouse (1:5000) immunoglobulin coupled to horseradish peroxidase (Dako, Courtaboeuf, France). Detection was carried out with an ECL Prime Western Blotting detection reagent (Amersham, Courtaboeuf, France) on a Fusion FX imager (Vilber Lourmat, Collegien, France), and analyses were performed using ImageJ software.
7
Immunofluorescence Staining of Oocyte Meiosis
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Progression to metaphase II (MII) was completed as described previously [17 (link),18 (link)]. At 16 or 22 h after beginning IVM, oocytes were denuded by vortexing then fixed in 4% [w/v] paraformaldehyde in Dulbecco’s phosphate-buffered saline (DPBS) for 15 min at room temperature. Oocytes were permeabilized with 1% [v/v] Triton X-100 in DPBS for 1 h at room temperature and blocked in 10% [v/v] donkey serum for 1 h at room temperature. Oocytes were incubated with a mix of 5 µg/mL mouse-ɑ-tubulin antibody and 5 µg/mL mouse anti-β-tubulin antibody (Sigma-Aldrich) either at room temperature for 1 h or overnight at 4 °C, then they were rinsed and incubated in Alexa Fluor 488 donkey anti-mouse (3.3 µg/mL) at room temperature for 1 h, and finally in 4′,6-diamidino-2-phenylindole (DAPI) (1 µg/mL) for 5 min at room temperature. Oocytes were placed into imaging droplets (DPBS containing 0.1% [w/v] Polyvinylpyrrolidone [PVP]) and imaged using an ECHO Revolve Epifluorescence Microscope with associated software (version R4) that includes the Z-stack software module (ECHO, San Diego, CA, USA). Oocytes were determined to have undergone meiosis II (MII) by detecting one polar body and a closely aligned cytoplasmic chromatin spindle.
8
PHOX2B Protein Expression Analysis
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3 × 105 IMR-32 cells were grown in a 6-multiwell plate and, 24 h and 48 h after treatments, they were washed with PBS 1×, centrifuged, and treated with RIPA buffer (Tris–HCl 50 mM pH 7.5, NaCl 150 mM, Triton-X 1%, SDS-20 0.1%, Na deoxycholate 1%, Protease Inhibitor mix 1×). Total cell lysates were quantified (Quick Start Bredford 1X Dye Reagent, BioRad) and equal amounts were electrophoresed on 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore). Chicken PHOX2B antibody [43 (link)], goat anti-chicken HRP antibody (Santa Cruz Biotechnology), mouse anti- β- tubulin antibody (Sigma-Aldrich), goat anti mouse HRP antibody (Dako) were used. Signals were detected using the ECL advance chemiluminescence reagent (Amersham). Images were acquired by UVITEC Alliance Mini HD9 Touch (Eppendorf) and quantification of the detected protein levels was performed by UVITEC NineAlliance 1D software.
9
Whole-Mount Oocyte Immunochemistry Protocol
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Whole-mount oocytes were fixed in a 2% paraformaldehyde solution supplemented with 0.1% Triton X-100 for 10 min at room temperature and blocked overnight in 1 mg/ml BSA in PBS, 0.01% Triton X-100. Oocytes were incubated overnight at 4°C with appropriate dilutions of the following antibodies: a mouse anti-BrdU antibody (cross-reactivity with Br-UTP established; 2 µg/ml; Sigma-Aldrich), a rabbit polyclonal anti-histone H4K8ac antibody (1:200; EMD Millipore), a mouse anti-H3K4me3 antibody (1:400; Abcam), a rabbit polyclonal anti-ATRX antibody (1:400; Santa Cruz Biotechnology), and a mouse anti–β-tubulin antibody (1:1,000; Sigma-Aldrich). Surface spread metaphase chromosome figures were prepared as described previously (Baumann et al., 2010 (link)). Immunochemistry was conducted by incubation with the following antibodies for 2 h at room temperature: rabbit polyclonal antibodies against histone H4K16ac (1:200; EMD Millipore), H4K20me (1:400; Abcam), H3S10ph (1:1,000; EMD Millipore), H4K5ac (1:200), and Smc4 (1:400) as well as with a human anti–Ana-Centromere C antibody (1:400, Cortex Biochem, Inc.). After several washes in PBS/BSA blocking medium, oocytes were exposed to 1:1,000 dilutions of the appropriate Alexa Fluor 488 or 555 secondary antibodies for 1 h at room temperature followed by DNA counterstaining and mounting in antifading medium.
10
Western Blot Analysis of Patched and Erk1/2
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Total RIPA extracts from cells or tumor homogenates were prepared. Protein concentrations were determined by the DC Protein Assay (Bio-Rad, Marnes-la-Coquette, France). Samples (50 to 80 µg) were separated on SDS-PAGE and transferred to nitrocellulose membranes (Amersham, Bath, UK) using standard techniques. After 1 h at room temperature in blocking buffer (20 mmol/L Tris-HCl pH 7.5, 45 mmol/L NaCl, 0.1% Tween-20, and 5% non-fat milk), nitrocellulose membranes were incubated overnight at 4 °C with rabbit anti-Patched antibody (Abcam ab53715; 1/1000), rabbit anti-Phospho-Erk1/2 antibody (Cell Signaling Technology (Leiden, The Netherlands); 1/1000), rabbit anti-Erk1/2 antibody (Cell Signaling Technology; 1/1000), or mouse anti-β-tubulin antibody (Sigma; 1/1000). After 3 washes, membranes were incubated for 45 min with anti-rabbit (1:2000) or anti-mouse (1:5000) immunoglobulin coupled to horseradish peroxidase (Dako-Agilent, Santa Clara, CA, USA). Detection was carried out with an ECL Prime Western blotting detection reagent (Amersham) on a Fusion FX imager (Vilber Lourmat, Collegien, France), and analyses were performed using ImageJ software.
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