Cells were lysed using RIPA lysis buffer (10 mM Tris-HCl [pH 7.4], 0.1% SDS, 1% sodium deoxycholate, 0.15 M NaCl, 1 mM EDTA, and 1% Triton X-100). Lysates were collected and cleared by centrifugation, and the protein concentration was quantified using the bicinchoninic acid method. Protein samples were electrophoresed by 12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (0.45 µm, Millipore). The membranes were blocked with 5% skim milk, and then incubated overnight at 4°C with the primary antibody (anti-YB-1, 1:1,000, Abcam; anti-MDM2, 1:1,000, Abcam). The membranes were then washed three times with Tris buffered saline with tween and incubated with horseradish peroxidase conjugated goat antirabbit secondary antibody (Beyotime) for 2 hours at room temperature. The bands of all proteins were normalized to the intensity of corresponding bands for β-actin. Protein expression was visualized with a chemiluminescence ECL kit (Beyotime).
Anti yb 1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-YB-1 is a primary antibody that recognizes the Y-box binding protein 1 (YB-1). YB-1 is a multifunctional protein involved in various cellular processes such as transcription, translation, and DNA repair. This antibody can be used to detect and study the expression and localization of YB-1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti mdm2, by Abcam (2 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Beyotime (1 mentions)
Ecl chemiluminescence kit, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Axio observer, by Zeiss (1 mentions)
Anti e cadherin, by BD (1 mentions)
Ez magna rip kit, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Taqman primer, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Geneamp 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence system, by PerkinElmer (1 mentions)
Anti α sma, by Merck Group (1 mentions)
Anti p16, by Abcam (1 mentions)
Imager 600, by GE Healthcare (1 mentions)
Normal donkey serum (nds), by Merck Group (1 mentions)
Anti g3bp, by Abcam (1 mentions)
Anti gfp, by Proteintech (1 mentions)
11 protocols using anti yb 1
1
Western Blot Analysis of Protein Expression
2
Immunofluorescence Analysis of Cell Markers
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IF assays were performed as previously described in [68 (link),69 (link)]. Briefly, treated and control HaCaT cells were seeded on glass coverslips at a density of 1.5 × 105 cells/well in 24-well dishes, fixed with 3.7% PFA, and permeabilized with 0.5% Triton X-100. After blocking with 3% BSA, cells were incubated with primary antibody (E-cadherin and YB-1) for 1 h at RT followed by incubation with Alexa-Fluor conjugated secondary antibodies for 1 h in the dark. To visualize the actin cytoskeleton, cells were stained with TRITC-conjugated phalloidin. The cells were counterstained with DAPI for the visualization of the nucleus. Images were taken with a Zeiss confocal laser-scanning microscope Axio Observer. A x40 objective was used and image analysis was performed using ImageJ. All images were taken with the same setting. Image processing and analysis were performed with Fiji (ImageJ version 2.0) software. The stress granules formation experiment was performed as described in [70 (link)] and images were acquired using a Nikon TE Eclipse 2000. Antibodies of anti-YB-1 (12148 Abcam, Cambridge, UK), anti-E-Cadherin (610181 BD Transduction Laboratories™, MA, USA), Alexa Fluor 488 anti-rabbit and anti-mouse (Thermo-Fisher Scientific, Waltham, MA, USA), and DAPI (Sigma-Aldrich, Saint Louis, MO, USA) were used.
3
RNA Immunoprecipitation Assay
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The RIP experiment was performed using the EZ-Magna RIP kit (Millipore, USA) according to the manufacturer's instructions. For each RIP assay, about 1 × 107 KGN, COV434 or SVOG cells were lysed in RIP lysis buffer supplemented with RNase inhibitor and protease inhibitor cocktail. After one freeze-thaw cycle, cell lysates were immunoprecipitated at 4°C overnight with anti-YB1 (Abcam, USA) or anti-ILF2 antibody (Abcam, USA). A homologous IgG was used as the isotype control. The co-precipitated RNA was extracted using TRIzol™ reagent according to the manufacturer's protocol. Then purified RNA was reverse-transcribed and subjected to qPCR analysis. Proteins isolated from the magnetic beads were detected using immunoblotting analysis. The details of antibodies are listed in Supplementary Table S3 .
4
Quantitative Proteomic and Transcriptomic Analysis
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The antibodies used in this study were as follows: anti- TERT (MBL, Nagoya, Japan), anti-p16, anti-YB-1 (Abcam, Cambridge, MA, USA), YB-1S102p (Cell signaling Technology, Beverly, Massachusetts, USA), anti-p21-HRP (Santa Cruz, CA, USA), anti-αSMA, anti-β-actin and anti-GAPDH (Sigma, St. Louis). Proteins were visualized using an enhanced chemiluminescence system (Perkin Elmer, Waltham, MA, USA) and scanned for quantitative analysis using an Imager 600 with ImageQuant TL software (GE Healthcare, Chicago, IL, USA).
Taqman primers (Thermo Fisher Scientific) were used for qPCR analysis of human and mouse TERT, αSMA, YB-1, p16, p21, IL6, SIRT1 and 18s rRNA. One-step RT-PCR was performed as before [60 (link)] using a GeneAmp 7500 sequence detection system (Applied Biosystems, Rockford, IL). Results were expressed as 2−ΔΔCT using the indicated control group as calibrator and 18srRNA as reference [61 (link)].
Taqman primers (Thermo Fisher Scientific) were used for qPCR analysis of human and mouse TERT, αSMA, YB-1, p16, p21, IL6, SIRT1 and 18s rRNA. One-step RT-PCR was performed as before [60 (link)] using a GeneAmp 7500 sequence detection system (Applied Biosystems, Rockford, IL). Results were expressed as 2−ΔΔCT using the indicated control group as calibrator and 18srRNA as reference [61 (link)].
5
Immunofluorescence Staining of Cellular Stress Granules
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Cells were fixed with 4% paraformaldehyde (4% PFA—Polysciences Inc., Hirschberg and der Bergstrasse, Germany; catalog number: 2878-55-4) in GibcoTM Dulbecco’s Phosphate-Buffered Saline (DPBS—Thermo Fisher Scientific (Brussels, Belgium), catalog number: 14190250) for 15 min and rinsed three times with DPBS. In total, 5 % normal donkey serum (NDS, Sigma, Machelen, Belgium; catalog number: D9663) in 0.1% Triton X-100 in DPBS was used for blocking at room temperature for 1 h. Primary antibodies were diluted in 2% NDS in 0.1% DPBS Triton and incubated overnight at 4 °C. The following primary antibodies were used: anti-G3BP (1/250, catalog number: ab56574), anti-Yb1 (1/500, catalog number: ab76149) and anti-importin-β1 (1/1000, catalog number: ab2811) all provided by Abcam (Cambridge, UK). The cells were subsequently washed with DPBS and incubated with appropriate secondary antibodies (1:2500; Thermo Fisher Scientific, Brussels, Belgium). Nuclei were stained with Hoechst (NucBlue Live ReadyProbesTM Reagent Hoechst 33342, Thermo Fisher Scientific, Brussels, Belgium; catalog number: R37605) and images were taken under SP8 confocal microscope (64x; Leica, Wetzlar, Germany; model: SP8 MDi8) excitation lines at 405, 488, 555 and 647 nm.
6
Western Blot Analysis of Protein Interactions
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The proteins were extracted in RIPA buffer (Pierce, Rockford, IL, USA) with 1% PMSF and boiled for 8–10 min, and separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then transferring protein to a PVDF membrane, blocking it with 5% skim milk for 3 h, and incubating with different primary antibodies and secondary antibodies successively. The primary antibodies used in WB included anti-Flag (Cell Signaling #14793), anti-Ub (Santa Cruz #F2819), anti-SIAH1 (Abcam ab2237), anti-YB-1 (Abcam ab12148), anti-GPADH (Cell Signaling D16H11), anti-GFP (Proteintech 66002-1-Ig).
7
Immunofluorescence Staining of Cellular Proteins
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Cells were plated on coverslips, and after 24 h of co-culturing, cells were fixed with 4% paraformaldehyde for 20 min and incubated with 10 mM NH4Cl for 10 min. The subsequent procedures were performed as previously described.8 (link) We used anti-Pgp (clone UIC2; Coulter), anti-Yb-1 (Abcam) and anti-NF-κB primary antibodies and Alexa 488-conjugated goat anti-rabbit IgG or Alexa 594-conjugated goat anti-mouse IgG secondary antibodies (Molecular Probes, Eugene, OR, USA). Images were acquired with the NIS-Elements F2.30 software, using an Eclipse E200 Nikon microscope connected to a Digital Sight system.
8
Co-immunoprecipitation of Protein Complexes
9
Immunohistochemical Analysis of YB-1 and MDM2 in Glioma
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Immunohistochemistry was performed to detect YB-1 and MDM2. Briefly, glioma tissues and cancer-free tissues were fixed using 10% buffered formalin and embedded using paraffin. The deparaffinized tissue sections were transferred to heat-induced epitope retrieval for 10 minutes and then incubated with the primary antibodies (anti-YB-1, 1:5,000, Abcam; anti-MDM2, 1:50, Abcam) at 37°C for 2 hours. Biotinylated secondary antibodies were applied to incubate tissue sections for 30 minutes. The stained cells in ten fields were counted under the microscope (Olympus).
10
Western Blot Analysis of Cellular Signaling
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Total cell lysates as well as nuclear and cytoplasmic enriched fractions were generated and used in Western Blot analysis as described earlier [9 (link)]. The primary antibodies applied were as follows: anti-PT202/Y204-ERK1/2, anti-ERK1/2, anti-PT359/S363-RSK, anti-RSK1/2/3, anti-PS102-YB-1, anti-PS112-Bad, anti-Bad, anti-PS473-AKT, anti-AKT, anti-caspase 3, anti-cleaved caspase 3, anti-cleaved PARP, anti-GAPDH, anti-Tubulinα/β (all Cell Signaling Technology); anti-LaminB (Santa Cruz Biotechnology); anti-YB-1 (Abcam). Immunodetection was carried out as described previously [9 (link)].
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