Total protein was extracted from tissues or cells. Protein lysates (50 μg/lane) were separated by 10% sodium lauryl sulfate-polyacrylamide gels (SDS-PAGE) and transferred onto a nitrocellulose membrane (Sigma-Aldrich; Merck KGaA). The membranes were blocked in 5% nonfat milk for 1 hour at room temperature and incubated at 4 °C overnight with a primary antibody: anti-LUM, anti-ERK, anti-p-ERK, anti-MEK1/2, anti-p-MEK1/2 and anti-MMP-2 (Abcam, Cambridge, MA), anti-Cyclin D1 (BOSTER, Wuhan, China). β-actin (Abcam, Cambridge, MA) was used as an internal control. After incubation with the secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 hour at room temperature, the signals were visualized using the LI-COR Image Studio lite imaging system.
Anti cyclin d1
Manufactured by Boster Bio
Sourced in China, United States
Anti-Cyclin D1 is a lab equipment product that is used to detect the presence and quantify the levels of Cyclin D1 protein in biological samples. Cyclin D1 is a crucial regulator of the cell cycle, and its expression is often altered in various disease states, including cancer. This product provides a reliable method for researchers to measure Cyclin D1 levels, which can help in the study of cell proliferation and the development of therapeutic interventions.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti p erk, by Abcam (1 mentions)
Anti erk, by Abcam (1 mentions)
Anti pmek1 2, by Abcam (1 mentions)
Anti mek1 2, by Abcam (1 mentions)
Image studio lite imaging system, by LI COR (1 mentions)
β actin, by Abcam (1 mentions)
Anti mmp2, by Abcam (1 mentions)
Enhanced chemiluminescence detection kit, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti shh, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti akt, by Bioss Antibodies (1 mentions)
Anti pakt, by Bioss Antibodies (1 mentions)
Anti cyclinb1, by Boster Bio (1 mentions)
Anti smo, by Santa Cruz Biotechnology (1 mentions)
Anti gli1, by Santa Cruz Biotechnology (1 mentions)
Phenylmethanesulfonyl fluoride, by Beyotime (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Beyotime (1 mentions)
5 protocols using anti cyclin d1
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Hedgehog Signaling
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Proteins were extracted using radio immunoprecipitation assay lysis buffer with 1% phenylmethanesulfonyl fluoride (Beyotime). After measuring protein concentrations with a BCA protein assay kit (Beyotime), 30 μg proteins from each group were separated by SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, United States). Following blockade with 5% skim milk or 1% bovine serum albumin, the membranes were incubated with anti-cyclinB1, anti-cyclinD1 (1: 400; Boster), anti-PTCH (1: 500; Novus Biologicals, Littleton, CO, United States), anti-Smo (1: 100; Santa Cruz, Dallas, TX, United States), anti-Gli1 (1: 200; Santa Cruz), anti-AKT, anti-p-AKT (1: 500; Bioss, Beijing, China), anti-Shh (1: 1000; Abcam, Cambridge, United Kingdom) or anti-β-actin (1: 1000; Santa Cruz) antibodies at 4°C overnight. The membranes were rinsed in tris buffered saline with Tween (TBST) and incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1: 5000; Beyotime) at 37°C for 45 min. Thereafter, the membranes were rinsed in TBST and visualized with an enhanced chemiluminescence detection kit (Beyotime). Relative protein levels were calculated using β-actin as the internal reference.
3
Western Blot Analysis of Key Proteins
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The western blotting protocols followed previous study (Wei et al., 2016 (link)). The primary antibodies used included: anti-Dmrt1 (1∶500; Santa Cruz Biotechnology, USA), anti-TLR2 (1∶200; Santa Cruz Biotechnology, USA), anti-TLR5 (1∶200; Santa Cruz Biotechnology, USA), anti-TLR4 (1∶200; Santa Cruz Biotechnology, USA), anti-NF-κB (1∶200; Santa Cruz Biotechnology, USA), anti-TNFα (1∶100; Proteintech Group, China), anti-IL-6 (1∶100; Proteintech Group, China), anti-PCNA (1∶200; Boster, China), anti-inositol-requiring enzyme-1 (IRE1) (Biosynthesis Biotechnology, China), anti-Chop (Biosynthesis Biotechnology, China), anti-p53 (1∶200; Wanlei Biotechnology, China), anti-cyclin-D1 (1∶200; Boster, China), anti-Plzf (1∶300; Sino Biological, China), anti-caspase-3 (Biosynthesis Biotechnology, China), and anti-GAPDH (Tianjin Sungene Biotech, China). Band intensities from three independent experiments were determined by densitometry using NIH ImageJ and the significance of differences was determined by Student’s t-test.
4
Protein Expression Analysis in Lung Cancer Cells
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Radioimmunoprecipitation (RIPA) lysis buffer (Beyotime) was used to extract total protein from A549 and H1299 cells. The same amount protein was subjected to sodium dodecyl‐sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gel and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Beyotime). The membrane was blocked with 5% skimmed milk and incubated with primary antibodies including anti‐NOVA2 (1:2000, Boster), anti‐β‐catenin (1:1500, Boster), anti‐c‐Myc (1:2000, Boster), anti‐CyclinD1 (1:1000, Boster), or anti‐GAPDH (1:2000, Boster). After the membrane was hatched with secondary antibody (1:10,000, Boster), the protein blots were visualized by Ultra Sensitive ECL Chemiluminescence Substrate (Boster). The relative protein expression was analyzed using ImageJ software (National Institutes of Health).
5
Protein Extraction and Western Blot Analysis
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Proteins were extracted using NP40 buffer (Beyotime Institute of Biotechnology, China) supplemented with protease inhibitor phenylmethanesulfonyl fluoride (PMSF; Roche, Switzerland). The protein concentrations were calculated using the bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, China). Equivalent amounts of 30 µg protein samples were isolated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, western blot analysis was performed following a standard procedure with the primary antibodies: anti-p21 (1:1000; Cell Signaling Technology, USA), anti-cyclin D1 (1:500; Boster, China), anti-CDK4 (1:500; Boster), anti-CDK6 (1:500; Boster), and anti-E-cadherin (1:1000; Cell Signaling Technology). The anti-β-Actin (Boster) was used as an internal control at 1:500 dilution. And then, protein bands were incubated with secondary antibodies (1:5000) conjugated with horseradish peroxidase (HRP) and visualized using the enhanced chemiluminescence (ECL) assay kit.
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