After cell lysis, bicinchoninic acid (BCA) (Abbkine, CA, USA) method was used to detect the protein concentration, and 30 μL protein was separated using 12% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Then the protein was transferred to a Polyvinylidene Fluoride (PVDF) membrane (Millipore, MA, USA), sealed with 5% skim milk powder for 2 h, and added with cyclin D1, c-jun, CD44, c-Met and β-actin primary antibodies purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Dilution ratio: 1: 1000, AF1183, AF1612, AF0105, AF1432, AF0003), for overnight incubation at 4°C. Then TBST buffer was applied for three rinses of the membrane, 10min each, followed by a 3-minute incubation with the corresponding horseradish peroxidase-labeled goat anti-rabbit (HRP)-labeled secondary antibody (Beyotime Biotechnology Co., Ltd., Shanghai, China, dilution ratio: 1:1000, A0208). The membrane was finally developed by ECL (Millipore, Germany), and the grayscale values were analyzed using Quantity One 1-D software.
Cyclin d1
Manufactured by Beyotime
Sourced in China, United States
Cyclin D1 is a protein that plays a crucial role in the regulation of the cell cycle. It is involved in the transition from the G1 phase to the S phase, which is the stage where the cell's DNA is replicated. Cyclin D1 acts as a regulatory subunit of the cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6) enzymes, which are essential for the progression of the cell cycle.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by ZSGB-BIO (2 mentions)
C myc, by Proteintech (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by ZSGB-BIO (2 mentions)
Af0003, by Beyotime (1 mentions)
β actin primary antibodies, by Beyotime (1 mentions)
Af1612, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Abs130868, by Absin (1 mentions)
Trans blot turbo mini pvdf transfer pack, by Bio-Rad (1 mentions)
Image pro plus software v 6.0, by Media Cybernetics (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Cell lysis buffer for western and ip, by Beyotime (1 mentions)
Protein free rapid blocking buffer, by Ipsen (1 mentions)
C myc, by Beyotime (1 mentions)
Immobilion p, by Merck Group (1 mentions)
β catenin, by Proteintech (1 mentions)
Gel doc xr system, by Bio-Rad (1 mentions)
β actin, by Beyotime (1 mentions)
Bcl 2, by Boster Bio (1 mentions)
12 protocols using cyclin d1
1
Protein Expression Analysis Protocol
2
Western Blot Analysis of PI3K-AKT Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed and loaded onto 10% precast polyacrylamide gels (Mini-PROTEAN TGX Precast Gels; Bio-Rad, Hercules, CA, USA); they were then blotted onto polyvinylidene difluoride (PVDF) membranes (Trans-Blot Turbo Mini PVDF Transfer Packs; Bio-Rad). After the membranes had been blocked blocking Tween 20 in PBS containing 5% non-fat dry milk, they were incubated with primary antibodies against p-PI3K (abs130868; Absin Bioscience, Inc., Shanghai, China), p-AKT (4060S; CST, Danvers, MA, USA), FOXO (2880T; CST), and Cyclin D1 (AF1183; Beyotime) at 4°C overnight. Membranes were incubated with a horseradish peroxidase–onjugated secondary anti-rabbit (WB0177) and IgG (WB0176), as appropriate. Relative expression was determined using Image-Pro Plus software (v. 6.0; Media Cybernetics, Rockville, MD, USA).
3
Western Blot Analysis of Protein Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each group of cells was lysed and denatured using Cell Lysis Buffer for Western and IP (Beyotime Institute of Biotechnology), and protein quantification was performed by the BCA method. A total of 25 µg of protein sample per lane was denatured and electrophoresed using 10% SDS-PAGE, followed by semi-dry transfer of protein onto a PVDF membrane (Immobilion-P; EMD Millipore) and protein blocking with Protein Free Rapid Blocking Buffer (Epizyme) at room temperature for 30 min. The membrane was then incubated with the following primary antibodies at 4°C for 15 h: GAPDH (1:2,000 dilution; cat. no. AF1186; Beyotime Institute of Biotechnology), β-actin (1:2,000 dilution; cat. no. AF1186; Beyotime Institute of Biotechnology), RRM2 (1:1,000 dilution; cat. no. 11661-1-AP; Proteintech), β-catenin (1:1,000 dilution; cat. no. AC106; Beyotime Institute of Biotechnology), cyclin D1 (1:2,000 dilution; cat. no. AF0126; Beyotime Institute of Biotechnology) and c-MYC (1:2,000 dilution; cat. no. 10828-1-AP; Proteintech). Subsequently, it was incubated with HRP-labeled Goat Anti-Rabbit IgG (1:1,000 dilution; cat. no. A0208; Beyotime Institute of Biotechnology) for 1 h at room temperature and final visualization was performed using a GelDoc XR System (Bio-Rad Laboratories, Inc.). The experimental results were analyzed in grayscale using Image J (v1.8.0; National Institutes of Health).
4
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lung tissues or treated A549 cells were harvested, and the whole proteins were extracted using a mixture of 100 µl RIPA lysate, 2 µl protease inhibitor (50×), and 1 µl 100 mM phenylmethanesulfonyl fluoride (PMSF, 100 mM). The protein concentration was determined using the bicinchoninic acid (BCA) method. Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes. Membranes were blocked in 5% non-fat milk in TBST for 1 h at 37°C and probed with primary antibodies overnight at 4°C. After three washes, the membranes were incubated with the corresponding secondary antibodies for 1 h at 37°C. Signals were detected with ECL western blot detection reagents (Millipore, Billerica, MA, USA) using a direct chemiluminescent detection system (Fusion 182Fx5, Vilber, France). Protein expression was quantitated using ImageJ software (NIH, Bethesda, MD, USA). The primary antibodies used were: mTOR (Cell Signaling Technology, USA), STAT3, Bcl-2 (BOSTER Biological Technology, China), cyclin D1 (Beyotime, Shanghai, China), β-actin (ZSGB-BIO, China), and GRP78 (a kind gift from Prof. Deqiang Wang, Chongqing Medical University, Chongqing, China).
5
Protein Expression Analysis in Cells and Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from cells and GC tissues using radioimmunoprecipitation assay lysis buffer containing protease inhibitors. Approximately 20 μg total protein from each group was resolved via 12 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (80 V). The duration of electrophoresis was determined based on molecular weight. Subsequently, the resolved protein bands were transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA), which was blocked with 5 % skimmed milk powder for 2 h at 37 °C and then incubated with primary antibodies against APOBEC3B (1:1,000, Abcam); P53 (1:1,000, Abcam); MCM2 (1:1,000, Abcam); cyclin D1 (1:1,000, Beyotime); and GAPDH (1:1,000, Goodhere) overnight at 4 °C. This was followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:2,000, ZSGB-BIO) at 37 °C for 2 h. GAPDH was used as an internal control for determining relative protein expression. Protein bands were quantified using ImageJ software (NIH).
6
Proteomic Analysis of Cullin-1 Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MLN4924 was purchased from Med Chem Express. Antibodies (Abs) for immunoblotting were all obtained from Cell Signaling Technology, except Deptor (Proteintech Group), Cullin-1 (Abcam, Cambridge, MA), cyclin-D1 and cyclin-D3 (Beyotime Institute of Biotechnology). The HRP-linked secondary antibodies were purchased from Sigma-Aldrich. Antibodies for APC-CD11c, PE-CD80, PE-CD86 and FITC-CD4 were purchased from eBioscience. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was obtained from Invitrogen.
7
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell extracts were prepared by lysing cells in ice-cold RIPA buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4, 1 NP-40, 0.1 SDS and 0.5 % deoxycholic acid) containing the protease inhibitor PMSF (Beyotime). Proteins (40 μg) were separated on 10 % SDS PAGE and transferred to PVDF membranes, and the blots were incubated with antibodies against human TROP2 (1 μg/ml), cyclin D1 (1:1000; Beyotime), E-cadherin (1:50000; Abcam), vimentin (1:500; Abcam) and GAPDH (1:2000; GoodhereBiotech Co., Hangzhou, China) as the protein loading control. Experiments were carried out in triplicate and repeated between three to five times.
8
Immunohistochemical Analysis of Cellular Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin-embedded tissues were deparaffinized in xylene and rehydrated using a graded ethanol series. Endogenous peroxidase activity was blocked by immersion in 0.3% hydrogen peroxide in methanol; tissues were then incubated in 10% normal goat serum for 30 min. Subsequently, tissues were incubated for 30 min with antibodies to cytochrome c oxidase (DF7867, 1:100; Affinity BioReagents, Golden, CO, USA), Cyclin D1 (AF1183, 1:100; Beyotime Biotechnology, Shanghai, China), or FoxO1 (2880T, 1:100; Cell Signaling Technology, Beverly, MA, USA). Immunoreactivity was developed with diaminobenzidine (Dako, Glostrup, Denmark) for 5 min; tissues were then counterstained with hematoxylin. Tissues were viewed under a microscope (XSP-C204; Chongqing Optical Instrument Co., Ltd., Chongqing, China); three fields were randomly photographed at a magnification of ×200. The integrated optical density of each image was analyzed.
9
TLR4 Signaling Pathway Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cellular protein was isolated from cultured cells with a protein extraction solution (Beyotime, china). Proteins were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes at 0.35 A (1 h, 4°C) using a wet-blotting apparatus (Bio-Rad). Membranes were blocked with 5% skim milk in PBST for 2 hours at room temperature, and then incubated overnight at 4°C with the primary antibodies diluted in PBST as follows: TLR4 (1:500), PAK1 (1:1000), phosphorylated-PAK1 Thr 423 (1:500; Santa Cruz), β-Catenin (1:1000), P-β-Catenin Ser 675 (1:1000; Cell Signaling), C-myc (1:1500; Proteintech), Cyclin D1 (1:200; Beyotime) and GAPDH (1:10000; Bioworld). Membranes were then incubated with appropriate secondary antibodies for 1 h at room temperature. Blots were quantified by densitometry using Quantity One 4.5.0 software (Bio-Rad Laboratories, Inc.).
The IP assay was conducted as previously described using an IP Kit (Beyotime, China) [36 (link)]. SW480 cells were stimulated with Fn (F01) and a total cell lysate was obtained. SW480 cells treated with DMSO were used as a control. Total protein was immunoprecipitated using an anti-TLR4 antibody (Santa Cruz) complexed to protein A/G. The immunoprecipitated proteins were then analyzed by western blot with an antibody to detect P-PAK1 T423.
The IP assay was conducted as previously described using an IP Kit (Beyotime, China) [36 (link)]. SW480 cells were stimulated with Fn (F01) and a total cell lysate was obtained. SW480 cells treated with DMSO were used as a control. Total protein was immunoprecipitated using an anti-TLR4 antibody (Santa Cruz) complexed to protein A/G. The immunoprecipitated proteins were then analyzed by western blot with an antibody to detect P-PAK1 T423.
10
Evaluating Protein Expression in Gastric Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein from cells and gastric cancer tissues was extracted using radioimmunoprecipitation assay lysis buffer containing protease inhibitors. Protein samples (20 μg) were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (12% resolving gel) and then transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). Membranes were then blocked with 5% nonfat milk to block nonspecific binding for 3 h at room temperature and incubated with primary antibodies against claudin 1 (1 : 1000, Abcam, Cambridge, MA, USA), caspase-3 (1 : 1000, Abcam), Bcl-2 (1 : 1000, Proteintech, Rosemont, IL, USA), c-Myc (1 : 1000, Proteintech), cyclin D1 (1 : 1000, Beyotime, Shanghai, China), and GAPDH (1 : 1000, Goodhere, Hangzhou, China) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000, ZSGB-BIO, Beijing, China) at room temperature for 3 h. GAPDH was used as an internal control for the relative protein expression. Protein bands were quantified using ImageJ software (NIH).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!