Cells were washed twice with PBS and total protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with PMSF protein inhibitor (Beyotime Institute of Biotechnology). The protein concentration was quantified using a standard BCA protein assay and 30 µg of protein/lane was separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto PVDF membranes (MilliporeSigma) and blocked with 5% non-fat milk at 26˚C for 2 h. The membranes were then incubated overnight at 4˚C with the following primary antibodies: Rabbit anti-COPS7A (1:1,000; cat. no. ab124705; Abcam), rabbit anti-CDK2 (1:1,000; cat. no. ab32147; Abcam), rabbit anti-CDK4 (1:1,000; cat. no. ab108357; Abcam), rabbit anti-cyclin D2 (1:1,000; cat. no. ab230883; Abcam) and rabbit anti-GAPDH (1:2,500; cat. no. ab9485; Abcam). Following the primary antibody incubation, the membranes were incubated with a goat anti-rabbit IgG H&L secondary antibody (1:5,000; cat. no. ab6721; Abcam) at room temperature for 2 h according to the protocol. The protein bands were visualized using an ECL system (Thermo Fisher Scientific, Inc.). GAPDH was used as the internal loading control.
Rabbit anti cdk4
Manufactured by Abcam
Sourced in United Kingdom, United States
Rabbit anti-CDK4 is a primary antibody that recognizes the Cyclin-Dependent Kinase 4 (CDK4) protein. CDK4 is a member of the cyclin-dependent kinase family and plays a crucial role in cell cycle regulation.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Rabbit anti cdk2, by Abcam (2 mentions)
Rabbit anti cyclin d1, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gapdh, by Abcam (1 mentions)
Pmsf protein inhibitor, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Antifade mounting medium with 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions)
Rabbit anti gapdh, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Yeasen (1 mentions)
Rabbit anti cyclin d1, by Cell Signaling Technology (1 mentions)
Rabbit anti cyclin b1, by Cell Signaling Technology (1 mentions)
Mouse anti γh2ax, by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti p21, by Abcam (1 mentions)
Mouse anti p53, by Abcam (1 mentions)
4 protocols using rabbit anti cdk4
1
Western Blot Analysis of Cell Cycle Regulators
2
Investigating CED's Effects on NSCLC
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The human NSCLC cell line A549 (p53 wild-type) was provided by the Cell Bank of Chinese Academy of Sciences (Shanghai, China). CED (purity > 99.58%) was purchased from MedChemExpress (New Jersey, USA). CED was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and stored at -80°C.
Fetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA, USA). Mouse anti-microtubule-associated protein B-light chain 3 (LC3B) was purchased from Cell Signaling Technology (Billerica, USA). Rabbit anti-GAPDH, mouse anti-mTOR, and rabbit anti-p-mTOR were purchased from Invitrogen (California, USA). Rabbit anti-VEGFR2, rabbit anti-VEGFR3, rabbit anti-P38, rabbit anti-p-P38, rabbit anti-Erk1/2, and rabbit anti-p-Erk1/2 were acquired from GeneTex (Taiwan, China). Rabbit Anti-CDK4, rabbit anti-cyclin D1, rabbit anti-CDK2, and rabbit anti-cyclin E were purchased from Abcam (Cambridge, England).
Cell counting kit-8 (CCK-8) was obtained from Yeasen Biotech (Shanghai, China). Antifade mounting medium with 4′,6-diamidino-2-phenylindole was obtained from Vector Laboratories (Shanghai, China). Annexin V-FITC/PI double staining was purchased from Becton, Dickinson and Company (New Jersey, USA).
Fetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA, USA). Mouse anti-microtubule-associated protein B-light chain 3 (LC3B) was purchased from Cell Signaling Technology (Billerica, USA). Rabbit anti-GAPDH, mouse anti-mTOR, and rabbit anti-p-mTOR were purchased from Invitrogen (California, USA). Rabbit anti-VEGFR2, rabbit anti-VEGFR3, rabbit anti-P38, rabbit anti-p-P38, rabbit anti-Erk1/2, and rabbit anti-p-Erk1/2 were acquired from GeneTex (Taiwan, China). Rabbit Anti-CDK4, rabbit anti-cyclin D1, rabbit anti-CDK2, and rabbit anti-cyclin E were purchased from Abcam (Cambridge, England).
Cell counting kit-8 (CCK-8) was obtained from Yeasen Biotech (Shanghai, China). Antifade mounting medium with 4′,6-diamidino-2-phenylindole was obtained from Vector Laboratories (Shanghai, China). Annexin V-FITC/PI double staining was purchased from Becton, Dickinson and Company (New Jersey, USA).
3
TDCPP-induced cytotoxicity mechanisms
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TDCPP (CAS no. 13674-87-8, 95.6% purity) was purchased from Sigma-Aldrich (St. Louis, MO, USA), and was dissolved in dimethyl sulfoxide (DMSO) as a stock solution. The Cell Counting Kit-8 was obtained from Dojindo Co. (Kumamoto, Japan). Rabbit anti-Caspase-3, rabbit anti-CDC-2, rabbit anti-Cyclin B1, rabbit anti-Phospho-CDC-2, rabbit anti-Cyclin D1 antibodies were obtained from Cell Signaling Technology (Danvers, CO, USA), mouse anti-γ-H2AX and rabbit anti-CDK-4 antibodies were obtained from Abcam (Cambridge, MA, USA). Secondary horseradish-peroxidase (HRP)-conjugated antibodies and rabbit anti-β-actin antibodies were purchased from Santa Cruz (Dallas, TX, USA). Chemicals for sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) were obtained from Amresco (Solon, OH, USA).
4
Protein Extraction and Western Blot Analysis
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The total protein was extracted by using RIPA lysis buffer (100μl, Beyotime, Shanghai, China). Lysates were centrifuged on ice for 20 min, followed by supernatants collection. BCA assay was utilized to determine the protein concentration. Proteins (20μg) were loaded and electrophoresed by using SDS-PAGE at 90 V for 1.5h, which were subsequently transferred to PVDF membranes. The membranes were blocked for 1h in 5% skim milk and then washed with PBST. Afterwards, the membranes were cultured with primary antibodies at 4°C overnight, followed by addition of secondary antibodies for another one-hour incubation. The membranes were washed three times for 5 min with TBST buffer. Finally, proteins were visualized through the ECL plus system. The primary antibodies included rabbit anti-Ezrin, rabbit anti-CyclinD1, rabbit anti-CDK4, mouse anti-P53 and rabbit anti-P21 (diluted 1: 200, Abcam); the secondary antibodies incorporated Goat Anti-Rabbit IgG H&L and Goat Anti-Mouse IgG H&L antibodies. β-actin was used as an internal reference.
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