Western blot assay was carried out as previously described.24 Cells were washed with PBS and then lysed with RIPA lysis buffer (Solarbio, China) and protease inhibitors (Roche Applied Science, Indianapolis, Switzerland). The protein concentration was measured using the bicinchonininc acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were subjected to 10% SDS‐polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were subsequently blocked with 5% non‐fat milk for 2 hours and incubated with primary antibodies overnight at 4°C. The primary antibodies used were anti‐MGMT (1:500, Abcam, Cambridge, UK) and anti‐Survivin (1:1000, Abcam), anti‐β‐catenin, anti‐CD44, anti‐C‐Jun, anti‐C‐Myc, anti‐cyclinD1, anti‐LEF1, anti‐TCF1/TCF7, anti‐MMP7, anti‐Axin2, anti‐Met, anti‐PARP, anti‐caspase‐3, anti‐BAX, anti‐Bcl2, anti‐cleaved‐caspase‐3, anti‐E‐cadherin, anti‐N‐cadherin and anti‐Vimentin (these primary antibodies are all: 1:1000, Cell Signaling Technology, Boston, MA, USA), as well as anti‐GAPDH, and secondary antibodies were HRP‐conjugated goat anti‐mouse or goat anti‐rabbit IgG antibody (1:1000, Beyotime). All experiments were performed in triplicate.
Anti survivin
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-survivin is a lab equipment product that functions as an antibody. It is used for the detection and analysis of the survivin protein, which plays a role in cell division and apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Anti bcl 2, by Abcam (6 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Anti β actin, by Merck Group (4 mentions)
Anti c myc, by Abcam (4 mentions)
Anti β actin, by Abcam (4 mentions)
Anti gapdh, by Abcam (4 mentions)
Anti e cadherin, by Cell Signaling Technology (3 mentions)
Anti β catenin, by Abcam (3 mentions)
Anti cyclin d1, by Abcam (3 mentions)
Anti caspase 3, by Abcam (3 mentions)
Anti bcl xl, by Abcam (3 mentions)
Anti mcl 1, by Abcam (3 mentions)
Anti n cadherin, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Anti p akt, by Abcam (2 mentions)
Anti cleaved caspase 3, by Abcam (2 mentions)
Anti hif 1α, by Abcam (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti cleaved parp 1, by Cell Signaling Technology (2 mentions)
32 protocols using anti survivin
1
Western Blot Analysis of Protein Targets
2
Protein Expression and Regulation Analysis
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Proteins were extracted using NP40 lysis buffer (Beyotime Institute of Biotechnology) and the bicinchoninic acid assay was applied to determine protein concentration. Proteins (20 µg) were separated by 10% SDS-PAGE and then transferred onto a PVDF membrane (cat. no. FFP28; Beyotime Institute of Biotechnology). The membrane was blocked using 5% skimmed milk at room temperature for 60 min and was then incubated with the following rabbit anti-human primary antibodies at 4°C for 12 h: Anti-HIF-1α (1:500; cat. no. ab51608; Abcam); anti-survivin (1:5,000; cat. no. ab76424; Abcam); anti-cleaved caspase-3 (1:500; cat. no. ab2302; Abcam); anti-phosphorylated (p)-mTOR (1:1,000; cat. no. ab109268; Abcam); anti-mTOR (1:2,000; cat. no. ab2732; Abcam); anti-p-Akt (1:500; cat. no. ab38449; Abcam); anti-Akt (1:500; cat. no. ab8805; Abcam); anti-p-PI3K (1:1,000; cat. no. ab182651; Abcam); anti-PI3K (1:1,000; cat. no. ab191606; Abcam); and anti-GAPDH (1:2,500; cat. no. ab9485, Abcam). The membrane was then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5,000; cat. no. ab205718; Abcam) at room temperature for 1 h. The bands were visualized by enhanced chemiluminescence (EMD Millipore), and densitometry was performed using the Bio-Rad ChemiDoc system with Image Lab software version 6.0 (Bio-Rad Laboratories, Inc.).
3
Quantitative Western Blot Analysis
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Total protein was extracted using RIPA buffer, and the concentration was quantified using a BCA Protein Assay Kit (Genshare Biological, China). Equal amounts of protein were loaded onto 10% SDS-PAGE gels for electrophoresis, transferred onto the PVDF membrane, blocked by 5% skim milk, and incubated with the primary antibody overnight at 4°C, including anti-survivin (1:500; Abcam, United States), anti-TGFb1(1:100; Abcam, United States), anti-pSmad3 (1:1,500; Abcam, United States), anti-BMPR2 (1:1,500; Abcam, United States), anti-HIF1α(1:500; Abcam, United States), anti-P53 (1:1,000; Abcam, United States), anti-Kv1.2 (1:400; Sigma, United States), anti-Kv1.5 (1:200; Abcam, United States), anti-Kv2.1 (1:500; Sigma), and anti-GAPDH (1:1,000; Epitomics, United States). The membrane was washed and incubated with HRP-conjugated goat anti-rabbit IgG (1:4,000; Abcam) at room temperature for 1 h. The blots were visualized using the chemiluminescence system and quantized by Quantity One.
4
Immunohistochemical Analysis of Survivin and Ki67
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After heat-induced epitope retrieval, paraffin-embedded sections were incubated with 3% H2O2 and blocked for another 60 min with 3% normal serum. Sections were then incubated with primary antibodies overnight at 4°C. An Elite ABC Staining Kit and DAB Peroxidase Substrate Kit (Vector Laboratories, Inc., Burlingame, CA, USA) were used to visualize antibody binding, according to the manufacturer’s instructions. Anti-survivin (Abcam, Cambridge, UK) and anti-Ki67 (Cell Signaling Technology, Beverly, MA, USA) antibodies were used for IHC. ImageJ software was used to analyze IHC staining in randomly selected slides from four mice in each group (more than six representative fields in each slide were analyzed). Positive staining in each slide was scored and presented as relative expression level (the protein expression level in shNC mice was arbitrarily set at 1). Data are expressed as means ± SD.
5
Western Blot Analysis of Protein Expression
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Total proteins were extracted from the MM cells and xenograft tumors using an ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) containing the protease inhibitor cocktail (Roche Diagnostics) and quantified using the Bio-Rad Bradford Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). Then, proteins (30 μg per lane) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA). After the blockade of nonspecific signals in 5% skimmed milk, the membranes were incubated overnight at 4°C with primary antibodies including anti-GAPDH (1:1,000 dilution; Abcam, Cambridge, UK), anti-NOTCH1 (1:2,000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), anti-hes family bHLH transcription factor 1 (Hes-1) (1:500 dilution; Abcam), anti-Survivin (Abcam), or anti-Bcl-2 (1:1,000 dilution; Abcam). Next, the membranes were incubated with the corresponding horseradish peroxidase-labeled secondary antibody (1:1,000 dilution; Abcam). Finally, the protein bands were imaged using the Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) in the Bio-Rad ChemiDox XRS imaging system (Bio-Rad Laboratories). Relative quantitative analysis of protein signals was performed by Quantity One software Version 4.1.1 (Bio-Rad Laboratories).
6
Western Blot Analysis of EMT Markers
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Cells were lysed in lysis buffer. Equal amounts of total proteins were loaded onto 4-12% SDS–PAGE gels and transferred to PVDF membranes (GE Healthcare Life Sciences, NJ, USA). The membranes were blocked with 5% milk dissolved in TBS containing 0.02% Tween 20 and incubated overnight at 4°C with specific primary antibodies. The membranes were subsequently incubated with specific horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using a Fusion FX5 system ((Vilber Lourmat, France). The following primary antibodies were used: anti-cleaved caspase 3, anti-E-cadherin, anti-cleaved caspase 9, anti-cleaved PARP1 (Cell Signaling Technology), anti-SNAIL1, anti-Vimentin, anti-Twist, anti-Slug, anti-Zeb1, anti-Nanog, anti-Sox2, anti-CD44 (Santa Cruz), anti-N-cadherin and anti-Oct4 (BD Biosciences), anti-Survivin, anti-CD133 (Abcam), anti-ALDH (Avivasysbio), and anti-β-actin (Sigma).
7
Investigating Survivin and NF-κB Signaling
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The anti-Id1 antibody was purchased from Abcam; anti-survivin, and anti-NFκB/p65 antibodies were obtained from Cell Signaling Technology. BAY 11–7082 (the NFκB blocker), and YM155 (survivin blocker) were obtained from Santa Cruz Biotechnology.
8
Quantifying Protein Levels via Western Blot
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Western blot analysis was used to determine changes in protein levels, according to a previous study (14 (link)). The primary antibodies used were rabbit polyclonal anti-TROAP (Cat. #HPA044102; Sigma-Aldrich, St. Louis, MO, USA), anti-METTL14 (Cat. #ab220031; Abcam, Cambridge, MA, USA), anti-cyclin D1 (Cat. #ab16663; Abcam), anti-survivin (Cat. # ab76424; Abcam), anti-p-AKT (Cat. #ab38449; Abcam), anti-WTAP (Cat. #ab195380; Abcam), and polyclonal rabbit anti-TROAP (Cat. #13634-1-AP; Proteintech, Rosemont, IL 60018, USA). The Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA) was used for protein imaging.
9
Western Blot Analysis of Stem Cell Markers
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Proteins were lysed by radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Shanghai, P.R. China), separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred into polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After blocking with 5% skim milk at room temperature for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. After being incubated with corresponding secondary antibodies (1:5,000; Cell Signaling Technology, Beverly, MA, USA) at room temperature for 1 h, the enhanced chemiluminescence (ECL) system (Bio-Rad Laboratories) was used for detection. The primary antibodies used in our study were anti-octamer-binding transcription factor 4 (OCT4), anti-sex-determining region Y box 2 (SOX2), anti-NANOG, anti-epithelial (E)-cadherin, and anti-vimentin (Cell Signaling Technology); anti-α-catenin, anti-β-catenin, anti-fibronectin, anti-c-myc, and anti-survivin (Abcam, Cambridge, UK); and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Sigma-Aldrich).
10
Immunofluorescence Staining of Retinal Cells
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Immunofluorescence staining was conducted as previously described Wu et al. (2013) (link). The eyes and RGCs from different groups were fixed with 4% paraformaldehyde (PFA) for 2 h and 20 min at room temperature, respectively. Then, the eyes were cut into 8-μm-thick sections after graded dehydration with 20 and 30% sucrose solution and then stored at -80°C until use. Sections and RGCs were incubated in 0.1% Triton X-100 and 3% (w/v) bovine serum albumin (BSA) (Sigma–Aldrich, St. Louis, MO, USA) for 30 min sequentially at room temperature to prevent non-specific background. Then, sections and RGCs were incubated with rabbit anti-MANF (1:200, Abcam, Cambridge, MA, USA) and anti-survivin (1:200, Abcam, Cambridge, MA, USA) antibody at 4°C overnight, followed by successive incubations with fluorescein-conjugated goat anti-rabbit secondary antibody (1:400, Molecular Probes, USA) and Hoechst staining. Negative controls were routinely prepared by incubating the cells and retinal sections in normal buffered serum instead of the primary antibody. The stained sections and cells were visualized and imaged using confocal microscopy (Leica SP8, Hamburg, Germany).
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