Western immunoblots were run as described previously 21 (link). Primary antibodies and their sources were as follows. Anti-total p53, anti-cyclin B1, anti-Bax, anti-Bcl-2, anti-CDK1, anti-phospho-CDK1, anti-activated caspase 8, anti-activated caspase 9, and anti-activated caspase 3 were from Cell Signaling Technology. Anti-ZIKV Env monoclonal antibody was purchased from Zoongen Co., Ltd (Beijing, China). Anti-p21Cip1/Waf1 and anti-6×His tag was from Abcam. Anti-β-actin and the secondary antibodies horseradish peroxidase-conjugated anti-rabbit IgG and anti-rabbit IgG were from Sigma-Aldrich. Anti-goat IgG was from Santa Cruz Biotechnology.
Anti cdk1
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Anti-CDK1 is a primary antibody that recognizes the Cyclin-dependent kinase 1 (CDK1) protein. CDK1 is a key regulator of cell cycle progression and plays a critical role in the G2/M phase transition. The Anti-CDK1 antibody can be used to detect and quantify CDK1 expression in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti cyclin b1, by Cell Signaling Technology (7 mentions)
Anti cdk2, by Cell Signaling Technology (5 mentions)
Anti akt, by Cell Signaling Technology (5 mentions)
Anti bax, by Cell Signaling Technology (4 mentions)
Anti parp, by Cell Signaling Technology (4 mentions)
Anti bcl 2, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti caspase 3, by Cell Signaling Technology (3 mentions)
Anti cdk4, by Cell Signaling Technology (3 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti ccnb1, by Cell Signaling Technology (2 mentions)
Anti ccna2, by Cell Signaling Technology (2 mentions)
Anti p21, by Cell Signaling Technology (2 mentions)
Anti p53, by Cell Signaling Technology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti cdk6, by Cell Signaling Technology (2 mentions)
Anti caspase 9, by Cell Signaling Technology (2 mentions)
Anti survivin, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
24 protocols using anti cdk1
1
Comprehensive Immunoblotting Approach
2
Comprehensive Immunoblotting Analysis of Cell Signaling
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Immunoblotting was performed with standard procedure. The following antibodies were used in this study: anti-NANOG (#8822, Cell Signaling Technology, MA, USA), anti-TFAP2C (sc-12762, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GAPDH (#5174, Cell Signaling Technology, MA, USA), anti-cleaved caspase-3 (#9664, Cell Signaling Technology, MA, USA), anti-cleaved PARP1 (ab32064, abcam), anti-CDH1 (#14472, Cell Signaling Technology, MA, USA), anti-β-CATENIN (#8480, Cell Signaling Technology, MA, USA), anti-PCNA (#13110, Cell Signaling Technology, MA, USA), anti-CCND2 (#3741, Cell Signaling Technology, MA, USA), anti-CCNA2 (#91500, Cell Signaling Technology, MA, USA), anti-CCNB1 (#4135, Cell Signaling Technology, MA, USA), anti-CDK1 (#9116, Cell Signaling Technology, MA, USA), anti-CDK2 (#2546, Cell Signaling Technology, MA, USA), anti- CCND1 (#55506, Cell Signaling Technology, MA, USA), and anti-EPCAM (#93790, Cell Signaling Technology, MA, USA). Immunoblots were visualized on iBright CL1000 Imaging Systems (Thermo Fisher Scientific).
3
Western Blot Analysis of Cell Cycle Markers
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Cells after treatment were washed with cold PBS and lysed on ice for 10 min in cell lysis buffer (Beyotime, Shanghai, China) containing PMSF (FDbio-tech, Hangzhou, China) and protease inhibitor cocktail (Biotool, Huston, USA). Lysates collected and centrifuged at 12,000× g for 20 min. Protein concentrations quantified by BCA Protein Assay Kit (Thermo Fisher, MA, USA), and 20 µg total protein separated on SDS/PAGE gels and subsequently transferred to polyvinylidene difluoride membranes. The membranes were blocked in 5% nonfat milk at room temperature for 1 h. Afterward, the membranes were incubated overnight at 4 °C with primary antibodies for anti-cyclinD1, anti-cyclinE2, anti-cyclinB1, anti-CDK1, anti–phospho-histone H3 (Cell Signal Technology, MA, USA), and GAPDH (Abcam, MA, USA), and followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (anti-mouse IgG, HRP-linked antibody and anti-rabbit IgG, and HRP-linked antibody) (Cell Signal Technology, MA, USA) for 2 h at room temperature. The hybridization signals detected using an Enhanced Chemiluminescence Detection kit (Fdbio-tech, Hangzhou, China).
4
Cell Cycle Regulation and Apoptosis
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The fluorogenic probe 2′, 7′-dichlorofluorescin diacetate (DCFH-DA, D6883) was from Sigma. 5-bromo-2′-deoxyuridine (BrdU, 000103) was provided by Thermo Fisher Scientific. Anti-BrdU antibody (#5292S), anti-phospho-H3-S10 (#3377), anti-topoisomerase II α (#12286), anti-phospho-P53 (#9284), anti-P21(#2947), anti-CDK1(#9116), anti-phosphor-CDK1(Tyr15) (#9111), anti-Cyclin B1(#4138), anti-PARP (#9542) and anti-Caspase 3 (#9662) were purchased from Cell Signaling Technology, anti-P53 (#AP6266d) was from Abcepta. Anti-β-Tubulin and anti-GAPDH antibodies were from Beijing TransGen Biotech (Beijing, China). Prestained Protein Ladder (26616) and M-PER buffer were from Thermo Pierce. FITC Annexin V Apoptosis Detection Kit I (#556547) and PI/RNase Staining Buffer (550825) were from BD Biosciences, protease inhibitor and phosphatase inhibitor cocktails were from Roche and the PVDF membrane was from Millipore.
5
Western Blot Analysis of Islet Cell Proteins
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For westerns, isolated islets or INS-1 cells grown in six well plates were homogenized in RIPA buffer and resolved on SDS-PAGE. Primary antibodies used were rabbit anti-c-Myc, 1:1000 (#5605, Cell Signaling, Boston, MA) and mouse anti-Gapdh, 1:5000 (#sc-32233, Santa Cruz). Secondary antibodies were anti-rabbit IRDye 800CW (#827-08365, Odyssey) and anti-mouse IRDye 680LT (#827-11080, Odyssey). Western analysis for cell cycle proteins on islets isolated from mice was carried out using the following antibodies—anti-Cdk1, 1:1000 (#9112, Cell Signaling Technologies), anti-Cdk2, 1:500 (#163, Santa Cruz Biotechnology), anti-Cdk4, 1:1000 (#260, Santa Cruz Biotechnology), anti-Cdk6, 1:500 (#3126, Abcam Inc.), anti-Cyclin A, 1:500 (#4710, Sigma), anti-Cyclin D3, 1:500 (#28283, Abcam Inc.), anti-Cyclin E, 1:500 (#481, Santa Cruz Biotechnology), anti-tubulin, 1:2000 (Calbiochem) and anti-actin, 1:2000 (Sigma).7 (link).
6
Protein Expression Analysis by Western Blot
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Whole-cell lysates were obtained using PRO-PREP protein extraction solution (Intron Biotechnology, Gyeonggi-do, Korea). Proteins were separated by SDS-PAGE, and bands were transferred to nitrocellulose membranes (Pall Corporation, Pensacola, FL, USA). The membranes were then incubated with the following primary antibodies at 4°C overnight: anti-p53, anti-Idh2, anti-B-actin, anti-Cdk1, anti-Cdk2, anti-Cdk4, anti-Cdk6, anti-iNOS, anti-Caspase3, and anti-PARP (Cell signaling); anti-p16 and anti-p21 (Abcam, Cambridge, UK); anti-COX-2 (SantaCruz); and anti-Prx-SO3 (Ab Frontier, Seoul, Korea). After washing with buffer, the membranes were incubated with the appropriate secondary antibody (Thermo Scientific) at room temperature for 6 hours.
7
Protein Expression Analysis by Western Blot
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Harvested cells were washed twice with phosphate-buffered saline (PBS), and proteins were extracted using 1× loading lysis buffer. Equal amounts of protein, as measured by the BCA protein assay, were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and transferred on to a polyvinylidenedifluoride (PVDF) membrane (Immobilon P, Millipore). The membrane was sealed in 5% skim milk for 1 h, and then incubated with primary antibody for 2 h. After washing with TBST for three times, it was incubated with horseradish peroxidase-bound secondary antibody for 2 h. Anti-PARP, anti-Caspase-3, anti-Caspase-9, anti-cyto C, anti-Bcl-2, anti-Bax, anti-PI3K, anti-p-PI3K, anti-Akt, anti-p-Akt, anti-CDK1, anti-Cyclin B1, anti-Cyclin A, anti-p21, anti-p27, anti-GAPDH, HRP-conjugated goat anti-rabbit IgG, and HRP-conjugated rabbit anti-mouse IgG antibodies were purchased from Cell Signaling Technology. After cleaning with TBST, the Universal ECL immunoblotting chemiluminescence solution (Vazyme, China) was used to display the bands, and relative expression of protein was analyzed by ImageJ.
8
Immunoprecipitation of CDK1, p62, and Flag
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NP40 extracts (1–2 mg) were incubated with normal mouse or rabbit sera for 30 minutes and subsequently with protein G or A-sepharose beads (GE Healthcare), respectively, for 1 hour at 4 °C. After centrifugation, beads were discarded and supernatants incubated for 2 hours with anti-CDK1 (Cell Signaling Technology), anti-Flag (Sigma-Aldrich) or normal mouse (Santa Cruz Biotechnology) monoclonal antibodies or serum, or anti-p62 (Novus Biologicals) polyclonal antibody or normal rabbit (Santa Cruz Biotechnology) serum, followed by protein G or A-sepharose beads for 1 hour. Beads were washed and bound proteins were solubilized by the addition of SDS-sample buffer heated at 95 °C for 5 minutes.
9
MBIC Modulates Cell Signaling Proteins
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Cells were treated with MBIC in a dose-dependent manner for 24 h. Cells were harvested, lysed and applied for Western blot analysis as described previously [10 (link)]. The primary antibodies that were used in this study included: anti-p53, anti-survivin, anti-phospho-survivin (anti-p-survivin), anti-Cdk1, anti-cyclin B1, anti-Bax, anti-caspase-3, anti-caspase-7, anti-caspase-9 (1:1000) (Cell Signaling Technology, USA) and mouse anti-β-actin (1:40000) (Sigma-Aldrich, St. Louis, MO, USA) antibodies. Western blot images were quantified and processed by ImageJ software (NIH, USA). Each experiment was done in triplicate.
10
Regulation of p27 Phosphorylation by CDK1
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HeLa cells (1 × 106) were transfected with two separate PFKFB3 siRNA species for 48 h. Post transfection, cell lysates were collected and standardized for protein content across all samples. Cdk1 was immunoprecipitated from lysates using anti-Cdk1 (C19, Santa Cruz Biotechnology) with rotation at room temperature for 1 h. Pre washed protein-G-sepharose (Dynabeads, Invitrogen) then was added to the IP samples for an additional 30 min at room temperature and subsequently washed 3 × with binding buffer. Immunoprecipitates were resuspended in a kinase reaction mixture containing 5 μg recombinant p27, 0.5 mM cold ATP, and 0.2 μCi 33P-ATP and incubated at 30 °C for 10 min. Reactions were removed from the sepharose beads and stopped by addition of 2 × Laemmli buffer. Proteins were separated via SDS-PAGE, and 33P-p27 was visualized by exposure to a phosphorscreen. Subsequently, immunoprecipitated CDK1 was determined by direct addition of Laemmli buffer to the beads and subjected to SDS-PAGE and western blot analysis using anti-Cdk1 (Cell Signaling Technology).
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