Antibodies to α-tubulin (Millipore, 05-829); Erk (9102), phospho-Erk (9101), Ras (3965), Raf-1 (12552S), p-Raf-1/Ser259 (9421S), FGFR2 (23328S), phospho-FGFR (3471S), H3 (4499), p-H3Ser10 (9701), cyclins D1, D3, A, B, E (Cyclin Antibody Sampler Kit, 9869T); CDKs 4 and 6 (CDK Antibody Sampler Kit, 9868T); cleaved caspase 3/Asp175 (9661S) (Cell Signaling); GPC1 (GeneTex, GTX104557); GAPDH (SAB2100894), laminin α1 (SAB4501255), FGF2 (SAB2100814), FGF1 (SAB1405808), β-catenin (C2206) (Sigma); α1(XI) (Oncomatrix, IS002); α3(V)-NTD (see below) and α1(V)46 (link) were all diluted 1:1,000. All were rabbit polyclonal except for CDK4 and 6, and α-tubulin antibodies, which were mouse monoclonal. Uncropped, full scan versions of immunoblots can be found in Supplementary Figs 9–14 .
Cyclin antibody sampler kit
Manufactured by Cell Signaling Technology
Sourced in United States
The Cyclin Antibody Sampler Kit is a collection of antibodies that recognize various cyclin proteins. Cyclins are key regulators of the cell cycle and play a crucial role in cell division. This kit provides a convenient way to detect and analyze the expression of different cyclin proteins in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Fibroblast growth factor 2 (fgf2), by Merck Group (1 mentions)
Cleaved caspase 3 asp175 9661s, by Cell Signaling Technology (1 mentions)
β catenin c2206, by Merck Group (1 mentions)
Gapdh, by Merck Group (1 mentions)
Gapdh mab, by Cell Signaling Technology (1 mentions)
Ab32028, by Abcam (1 mentions)
Ab84400, by Abcam (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Autophagy antibody sampler kit, by Cell Signaling Technology (1 mentions)
Polybrene sc 134220, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine (dab), by Beyotime (1 mentions)
Incucyte zoom, by Sartorius (1 mentions)
Facscalibur, by BD (1 mentions)
α tubulin, by Merck Group (1 mentions)
Acetyl α tubulin, by Merck Group (1 mentions)
Cell cycle regulation antibody sampler kit, by Cell Signaling Technology (1 mentions)
6 protocols using cyclin antibody sampler kit
1
Antibody Profiling of Cell Signaling Pathways
2
Protein Expression Analysis Protocol
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Cells or tissues were lysed in RIPA buffer. The following primary antibodies were used to detect the proteins of interest: (1) cyclin antibodies (Cyclin Antibody Sampler Kit, Cell Signaling, Danvers, MA, USA); (2) mTOR mAb, 4E-BP1 mAb, p70 S6 kinase mAb, and GAPDH mAb (Cell Signaling, Danvers, MA,USA); (3) mouse GFAP mAb and rabbit DCX polyclonal antibody (Santa Cruz, Dallas, TX, USA). Protein bands were visualized and optical density was analyzed using a Biospectrum 500 imaging system (UVP, LLC, Upland, CA, USA).
3
Comprehensive Antibody Procurement Protocol
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The NUCKS antibody (12023–2-AP) was purchased from Proteintech (Wuhan, China). Polybrene (sc-134,220) was purchased from Santa Cruz Biotechnology (Shanghai, China). Antibodies against mammalian target of rapamycin (mTOR) (ab32028) and phosphor-S2448 mTOR (ab84400) were purchased from Abcam (Shanghai, China). The Cyclin Antibody Sampler Kit (no. 9869), apoptosis antibody (no. 5625, 9664, 3498), Phospho-p70 S6K antibody (no.9234) and Autophagy Antibody Sampler Kit (no.4445) were purchased from Cell Signaling Technology (Shanghai, China). The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (M5655), 5′-bromo-2-deoxyuridine (BrdU), and dimethyl sulfoxide (DMSO) (D5879) were obtained from Sigma-Aldrich. The tubulin antibody, 4′, 6-diamidino-2-phenylindole (DAPI) and 3, 3′-diaminobenzidine (DAB) were purchased from Beyotime (Shanghai, China). HRP goat anti-mouse and goat anti-rabbit antibodies were purchased from KPL, Inc. (Maryland, USA). Alexa Fluor 488 goat anti-rabbit IgG (H + L), Lipofectamine 2000, and puromycin (A1113803) were obtained from Life Technologies (Shanghai, China). The BrdU antibody (ab6326) was purchased from Abcam (Shanghai, China). All antibodies were diluted according to the instructions. Chloroquine and rapamycin were acquired from Must Bio-technology (Chengdu, China) and Beyotime (Shanghai, China), respectively.
4
Proteasome Inhibition and Cell Cycle Analysis
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Cell proliferation during drug treatment was visualized by live cell imaging using an Incucyte Zoom® instrument (Essen Bioscience, Ann Arbor MI, USA). MelJuSo UbG76V YFP cells were used as a reporter line for proteasome activity. Cells were treated with 250 nM b-AP15 and imaged every 10 minutes. Lineage tracing of cells was performed using these captures.
Flow cytometry was used to analyze cell cycle distribution. For determination of cell cycle HCT-116 cells were treated with b-AP15 (1μM), bortezomib (200nM), or DMSO as control. Cells were harvested by trypsinization then washed and fixed in 70% ice cold EtOH for 12 h. Cells were re-suspended in staining solution containing propidium iodide (50 μg/ml) and RNAse A (0.5 μg/ml) in PBS. Flow cytrometry was performed using the BD FACScalibur. Data was analyzed using ModFit software.
Protein levels of cyclins were analyzed using western blotting. HCT116 cells were treated with b-AP15 (0.5μM) for 6 hours. After gel electrophoresis proteins were detected with indicated antibodies at manufacturer’s recommended conditions (Cyclin Antibody Sampler Kit, Cell Signal Technology, Danvers MA, USA).
Flow cytometry was used to analyze cell cycle distribution. For determination of cell cycle HCT-116 cells were treated with b-AP15 (1μM), bortezomib (200nM), or DMSO as control. Cells were harvested by trypsinization then washed and fixed in 70% ice cold EtOH for 12 h. Cells were re-suspended in staining solution containing propidium iodide (50 μg/ml) and RNAse A (0.5 μg/ml) in PBS. Flow cytrometry was performed using the BD FACScalibur. Data was analyzed using ModFit software.
Protein levels of cyclins were analyzed using western blotting. HCT116 cells were treated with b-AP15 (0.5μM) for 6 hours. After gel electrophoresis proteins were detected with indicated antibodies at manufacturer’s recommended conditions (Cyclin Antibody Sampler Kit, Cell Signal Technology, Danvers MA, USA).
5
Protein Extraction and Immunoblotting
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Collected cells were lysed in radioimmunoprecipitation assay buffer (RIPA buffer) containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1% SDS, 0.25% sodium deoxycholate, 10 mM NaF, 1 mM Na3VO4, and protease inhibitor cocktail (Roche, Switzerland). Primary antibodies against acetyl-α-tubulin (Sigma), α-tubulin (Sigma), Cyclin Antibody Sampler Kit (Cell Signaling Technology, Danvers, MA, USA), and Acetyl-Histone H3 Sampler Kit (Cell Signaling Technology, USA) were used.
6
Immunoblotting for Cell Cycle Analysis
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Cell protein lysates were electrophoresed on 4% SDS-PAGE gels for FANCD2 analysis and 12% SDS-PAGE gels for cell-cycle markers. Proteins were transferred to PVDF membrane, blocked, and incubated with primary antibodies as previously described [19 (link)]. Table 1 summarizes detailed information for the antibodies used against FANCD2, nuclear matrix protein p84, and all antibodies for cell cycle analysis from cyclin antibody sampler kit (#9869, Cell Signaling, MA, USA) and cell cycle regulation antibody sampler kit (#9932, Cell Signaling).
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