The GC cell lines NUGC-3, MKN-1, MKN-45, HGC-27, and NUGC-4 were obtained from Shanghai Bioleaf Biotech Co., Ltd. (Shanghai, China). Cells were cultured in RPMI 1640 medium (Kino Biological and Pharmaceutical Technology Co., Ltd., Hangzhou, China) containing 10% foetal bovine serum (FBS, Gibco, Grand Island, USA) and 1% penicillin/streptomycin (Kino Co., Ltd., Hangzhou, China) in a humidified atmosphere containing 5% CO2/95% air at 37 °C. The antibodies anti-GPx4 (Cat No. ab125066), anti-CDK4 (Cat No. ab108357), anti-CDK4 (Cat No. ab124821), anti-Cyclin D1 (Cat No. ab16663), anti-Ferritin Heavy chain (Cat No. ab287968) and anti-TFRC (Cat No. ab214039) were purchased from Abcam (Cambridge, UK). Anti-LC3B (Cat# 2775) antibody was purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-NCOA4 (Cat. Sc-373739) was purchased from Santa Cruz Biotechnology (Texas, USA).
Anti cdk4
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-CDK4 is a primary antibody that recognizes the CDK4 (Cyclin-Dependent Kinase 4) protein. CDK4 is a key regulator of the cell cycle and plays a crucial role in the G1/S phase transition. This antibody can be used for applications such as Western blotting, immunoprecipitation, and immunohistochemistry to detect and study the expression and localization of CDK4 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cyclin d1, by Abcam (26 mentions)
Anti cdk6, by Abcam (14 mentions)
Anti gapdh, by Abcam (10 mentions)
Anti p21, by Abcam (6 mentions)
Anti cyclin e1, by Abcam (6 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Anti c myc, by Abcam (5 mentions)
Anti cdk2, by Abcam (5 mentions)
Bca protein assay kit, by Beyotime (5 mentions)
Anti β actin, by Abcam (5 mentions)
Anti β actin, by Merck Group (5 mentions)
Anti cleaved caspase 3, by Abcam (4 mentions)
Anti p53, by Abcam (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Anti cyclin d3, by Abcam (4 mentions)
Anti rb, by Abcam (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Anti vimentin, by Abcam (3 mentions)
Anti n cadherin, by Abcam (3 mentions)
Anti p27, by Abcam (3 mentions)
49 protocols using anti cdk4
1
Gastric Cancer Cell Line Cultivation
2
Ischemic Stroke Protein Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from the infarct side of cerebral cortex with RIPA lysis buffer (Beyotime Institute of Biotechnology). BCA Protein Assay reagent (Beyotime Institute of Biotechnology) was applied to analyze proteins concentration. Proteins (50 µg/lane) were separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. Subsequently, the membranes were blocked with 5% non-fat milk powder for 2 h at room temperature and incubated with primary antibodies, including anti-EphB4 (Abcam; cat. no. ab254300; 1:1,000), anti-ephrinB2 (Abcam; cat. no. ab75868; 1:500), anti-phosphorylated (p)-ABL proto-oncogene 1, non-receptor tyrosine kinase (ABL1; Cell Signaling Technology, Inc.; cat. no. 2864; 1:1,000), anti-ABL1 (Cell Signaling Technology, Inc.; cat. no. 2862; 1:1,000), anti-Cyclin D1 (Abcam; cat. no. ab134175; 1:2,000), anti-CDK4 (Abcam; cat. no. ab108355; 1:1,000) and β-actin (Abcam; cat. no. ab8227; 1:1,000) at 4°C overnight, followed by the horseradish peroxidase-labeled secondary antibodies (Abcam; cat. nos. ab205718 and ab205719; both 1:2,000) at 37°C for 1 h. The immunoreactive proteins were visualized using an ECL kit (MilliporeSigma) and the data were analyzed using ImageJ software (version 1.8.0; National Institutes of Health). The expression values were normalized against β-actin.
3
ASIC1a Regulation of Apoptosis Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The antibodies used: anti-ASIC1a, anti-Bcl-2, anti-cleaved-caspase3, anti-Bax, anti-c-Myc, anti-Cyclin D, anti-CDK4, anti-GSK3β, anti-p-GSK3β (Ser9), anti-β-catenin, anti-p-β-catenin (Ser33) were from Abcam (Cambridge, UK). Anti-GAPDH, anti-HA and anti-Lamin B were from Santa Cruz Biotechnique (Santa Cruz, USA). Dual luciferase reporter assay system was from Promega Corporation (Wisconsin, USA). Reporter constructs were generated by incorporating 8X lymphocyte enhancer-binding factor-T cell factor (LEF-TCF) consensus binding sites into the pGreenFire1 (pGF1) vector containing eGFP-T2A-lucifersase as the reporter (System Biosciences) [29 ]. cDNAs were cloned into mammalian expression vectors pCDNA6-CMV-V5/His (Invitrogen) or a modified pCDH1-EF1 vector (System Biosciences). We obtained from Addgene the pcDNA3-HA-Ub construct (no. 18712) [30 (link)]. Lenti-cas9 and Lenti-sgRNA were purchased from Genechem (Shanghai, CHN). Cells were firstly infected with Lenti-cas9 and selected by puromycin. The stable sub-lines were then infected with Lenti-sgRNA to specifically knockout target genes. The sgRNA used were sg-GFP: 5′- GGTGAACCGCATCGAGCTGA-3′; sg-ASIC1a: 5′- GACGAGACGTCCTTCGAAGC-3′. All other chemicals were from Sigma–Aldrich Corporation (San Luis, USA) unless otherwise stated.
ASIC1α-specific inhibitor PcTx1 was purchased from Abcam (ab120483, Cambridge, MA, USA).
ASIC1α-specific inhibitor PcTx1 was purchased from Abcam (ab120483, Cambridge, MA, USA).
4
Comprehensive Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins of cells were obtained using RIPA lysis buffer (#89900; Thermo Fisher Scientific, Waltham, MA, USA) along with protease inhibitor cocktail and phosphorylase inhibitor. The concentration of the protein was quantified using the Bicinchoninic Acid (BCA) method. In detail, 25 μg protein of each sample was loaded onto a gel for separation and transferred to Polyvinylidene Difluoride (PVDF) membranes for exposure. Antibodies used in this experiment were listed as follows: anti-KCNK9 (#ab85289; Abcam, Cambridge, UK), anti-KCNK3/TASK1 (#ab135883; Abcam), anti-GAPDH (#ab8245; Abcam), anti-Ki67 (#ab16667; Abcam), anti-E-cadherin (# 3195; Cell Signaling Technology [CST], Inc., Danvers, MA, USA), anti-N-cadherin (#13116; CST Inc.), anti-Bax (#5023; CST Inc.), anti-Bcl-2 (#15071; CST Inc.), anti-caspase3 (#9662; CST Inc.), anti-cleaved caspase-3 (#9654; CST Inc.), anti-PARP (#9532; CST Inc.), anti-cleaved PARP (#9185; CST Inc.), anti-beta catenin (#ab32572; Abcam), anti-c-Myc (#ab185656; Abcam), anti-P53 (#ab26; Abcam), anti-cyclin D1 (#ab16663; Abcam), anti-CDK6 (#ab124821; Abcam), anti-CDK4 (#ab108357; Abcam), anti-vimentin (#ab92547; Abcam), and anti-vimentin (#ab27568; Abcam).
5
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins of thecells were extracted by lysis buffer (Tris-HCl, PH 8.0, 400 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM Na pyrophosphate, 1% Triton X-100, 10% glycerol), with supplement of protease inhibitors (Roche, Indianapolis, IN). The protein concentration was determined by a BCA kit (Piece, Rockford, IL). Then protein was loaded onto 12% SDS-PAGE gel and was electronically transferred to PVDF membranes (Millipore, Billerica, MA). Primary and secondary antibodies were then incubated to the membranes. The HRP-conjugated secondary antibody (1:5000, Sigma-Aldrich) was incubated for 2 h atroom temperature. Protein signals were detected via ECL method. The primary antibodies used in this study includes anti-BRD4 (1:1000, Abcam), anti-c-Myc (1:1000, Abcam), anti-CyclinD1 (1:1000, Abcam), anti-CDK4 (1:1000, Abcam), anti-MMP2 (1:1000, Abcam), anti-MMP9 (1:1000, Abcam), anti-CtIP (1:1000, Abcam), anti-CD274 (1:1000, Abcam), anti-KRAS (1:500, Abcam), anti-E-cadherin (1:10000, Abcam), anti-N-cadherin (1:1000, Abcam), anti-Vimentin (1:3000, Abcam) and anti-GAPDH (1:1000, Abcam). All the primary antibodies were incubated for 4 h at 37 °C.
6
Protein Expression Analysis in ESCC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates of ESCC cells were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22-mm NC membranes (Sigma), and incubated with specific antibodies: anti-CDK6, anti-CDK4, anti-CDK2, anti-cyclinD1, anti-cyclinD3, anti-p27, anti-p21, anti-CDKN2C (Abcam, Shanghai, China), anti-EZH2, anti-EED, anti-SUZ12, anti-EZH1, and anti-β-actin (Cell Signaling Technology). The dilution ratio of the primary antibodies was 1:1,000, although anti-β-actin was diluted to 1:8,000 for Western blotting. Protein bands were visualized with Super Signal Chemiluminescence Substrate (Thermo Scientific) and β-actin was used as a control.
7
Protein Expression Analysis in Colorectal Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein from the treated and untreated HCT116 and LOVO cells was extracted by RIPA (Beyotime, Nantong, China). The protein lysates were separated by SDS-PAGE and then transferred onto PVDF membranes. ECL chromogenic substrate was used for densitometry quantification after incubating specific antibodies at 4 °C for 12 h. Protein detection was performed using rabbit monoclonal anti-CCND1 (Abcam, Cambridge, UK, ab134175), anti-CDK4 (Abcam, ab108357), anti-P21 (Abcam, ab109520), anti-casepase3 (CST, #14220), anti-PARP (Proteintech, Rosemont, IL, USA, 13371-1-AP), anti-PDCD4 (Abcam, ab80590), anti-Vimentin (CST, #5741), anti-N-cadherin (Abcam, ab76011), anti-E-cadherin (Abcam, 76011), anti-METTL3 (Proteintech, 15073-1-AP), anti-IGF2BP2 (Proteintech, 11601-1-AP),anti-FLAG (Proteintech, 20543-1-AP), anti-pAKT (CST, #4060), anti-AKT (CST, #9272), anti-PI3K (CST, #4249), anti-pPI3K (CST, #4228). GAPDH (Affinity, West Bridgeford, UK, AF7021) was applied as the internal control.
8
Western Blot Analysis of CDK Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from CRC cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) and quantified using a BCA protein assay kit (Beyotime Institute of Biotechnology). Proteins (40 µg per lane) were separated via 10% SDS-PAGE, transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.) and blocked with 5% skimmed milk at room temperature for 1 h. The membranes were then incubated at 4˚C overnight with the following primary antibodies: Anti-CDK2 (1:1,000; cat. no. ab32147; Abcam), anti-CDK4 (1:1,000; cat. no. ab32147; Abcam), anti-CDK6 (1:1,000; cat. no. ab124821; Abcam) and anti-β-actin (1:1,000; cat. no. ab8226; Abcam). Following the primary antibody incubation, the membranes were incubated with a HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5,000; cat. no. ab7090; Abcam) at room temperature for 1 h. Protein bands were visualized using an ECL kit (Thermo Fisher Scientific, Inc.). β-actin was used as the loading control. ImageJ software (version 6.0; National Institutes of Health) was used for densitometry.
9
Genetic Manipulation of Glioma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The paternal GL261 mouse glioma cell line (DSMZ, ACC802) was used to generate Egfr, Cdk4 and Egfr+Cdk4 overexpressing subpopulations by lentiviral transduction. Male sex of the cell line was confirmed by Y chromosome specific PCR. Murine Egfr or Cdk4 was cloned into pLenti-CMV-Puro or pLenti-CMV-Neo plasmid (Addgene) and lentiviral particles were produced according to Life Technologies protocols. After viral transduction GL261 cells were selected with 1.5 μg/ml puromycin and/or 300ug/ml neomycin for 4 days and oncogene overexpression was confirmed by western blotting using anti-Egfr (Cell Signaling, 2232S, 1:1000), anti-Cdk4 (Abcam, ab137675, 1:5000), and anti-GAPDH (Cell Signaling, 8884S, 1:1000). Lentiviral particles for expression of fluorescent proteins were prepared in the same way as described above using pLV-eGFP and pLV-mCherry vectors (Addgene). Cells overexpressing eGFP and/or mCherry were sorted using BD FACS Aria III (BD Biosciences) and overexpression of the oncogenes was again confirmed by Western blot analysis.
All cell lines were cultured DMEM media (Gibco) with 10% fetal bovine serum (FBS, Gibco) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, PenStrep, Gibco) at 37 °C in a humidified atmosphere with 5% CO2. Cells were routinely checked for mycoplasma contamination using Mycoplasma PCR Detection kit (Applied Biological Materials, G238).
All cell lines were cultured DMEM media (Gibco) with 10% fetal bovine serum (FBS, Gibco) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, PenStrep, Gibco) at 37 °C in a humidified atmosphere with 5% CO2. Cells were routinely checked for mycoplasma contamination using Mycoplasma PCR Detection kit (Applied Biological Materials, G238).
10
Western Blot Analysis of HDACI-Treated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment with HDACIs, the cells were lysed with lysis buffer (Cell Signaling, Danvers, MA) including a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Western blot analysis was performed as previously reported [40 (link)]. Antibodies used for immunodetection were anti-p21 (1:1,000, Abcam, Cambridge, MA), anti-p27 (1:500, Abcam), anti-CDK2 (1:500, Thermo Scientific, Rockford, IL), anti-CDK4 (1:200, Abcam), anti-acetyl H3 (K9 + K14 + K18 + K23 + K27, 1:1,000, Abcam), anti-acetyl H3 (K27,1:2,000, Abcam), anti-acetyl H4 (K5 + K8 + K12 + K16,1:2,000, Abcam) and anti-β-actin (1:10,000, Sigma-Aldrich).
After the blot was washed, it was incubated with a horseradish peroxidase-conjugated species-specific secondary antibody (1:5,000, Jackson Laboratory, West Grove, PA) for 1 h at room temperature. The blots were developed using a chemiluminescence detection system (Invitrogen, Carlsbad, CA). Band density was analyzed using NIH ImageJ software. Densitometric measurements were performed on individual immunoblot for each antibody tested, and the values indicate the protein level normalized to the corresponding β-actin levels.
After the blot was washed, it was incubated with a horseradish peroxidase-conjugated species-specific secondary antibody (1:5,000, Jackson Laboratory, West Grove, PA) for 1 h at room temperature. The blots were developed using a chemiluminescence detection system (Invitrogen, Carlsbad, CA). Band density was analyzed using NIH ImageJ software. Densitometric measurements were performed on individual immunoblot for each antibody tested, and the values indicate the protein level normalized to the corresponding β-actin levels.
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!